http://2013.igem.org/wiki/index.php?title=Special:Contributions/Idonnya&feed=atom&limit=50&target=Idonnya&year=&month=2013.igem.org - User contributions [en]2024-03-28T23:57:39ZFrom 2013.igem.orgMediaWiki 1.16.5http://2013.igem.org/Team:Paris_Bettencourt/Project/TargetTeam:Paris Bettencourt/Project/Target2013-10-29T03:48:54Z<p>Idonnya: </p>
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<h2>Background</h2><br />
<p>SirA is an essential gene in latent tuberculosis infections</p><br />
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<div class="results"><br />
<h2>Results</h2><br />
<ul><br />
<li>Produced an <i>E. coli</i> strain which relies upon mycobacterial sirA, fprA and fdxA genes to survive in M9 minimal media</li><br />
<li>Demonstrated that <i>E. coli</i> can survive with mycobacterial sulfite reduction pathway with Flux Balance Analysis</li><br />
<li>Located drug target sites on sirA as well as identified high structural similarity between cysI and sirA through structural anaylsis</li><br />
<li>Identified a potential anti-TB activity of Pyridoxine at high doses.</li><br />
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<p></p><br />
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<div class="biocriks"><br />
<h2>BioBricks</h2><br />
<ol><br />
<li><a href="http://parts.igem.org/Part:BBa_K1137000">BBa_K1137000 (SirA)</a></li><br />
<li><a href="http://parts.igem.org/Part:BBa_K1137001">BBa_K1137001 (FprA)</a></li> <br />
<li><a href="http://parts.igem.org/Part:BBa_K1137002">BBa_K1137002 (FdxA)</a></li><br />
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<div class="aims"><br />
<h2>Aims</h2><br />
<p>To perform a drug screen targeted at the sirA gene from mycobacteria</p><br />
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<a href="#Introduction"><br />
<div class="hlink"><br />
<h2>Skip to Introduction</h2><br />
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<a href="#Model"><br />
<div class="hlink"><br />
<h2>Skip to Modeling</h2><br />
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</a><br />
<a href="#Design"><br />
<div class="hlink"><br />
<h2>Skip to Design</h2><br />
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<a href="#Results"> <br />
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<h2>Skip to Results</h2><br />
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<div id="Introduction"></div><br />
<h2>Introduction</h2><br />
<div class="leftparagraph"><br />
<p> &nbsp;&nbsp;<br />
SirA is essential for <i>M. tuberculosis</i> persistence phenotype as sulfur containing amino acids are particularly sensitive to oxidative stress within the macrophage and must regularly be replaced <a href="#Reference">(Pinto <i>et al</i> 2007)</a>. Currently, there are no drug candidates that are known to specifically inhibit SirA and conventional drug screens involve do not provide information regarding the mechanism of drug action nor do compounds that inhibit exponential growth necessarily have an effect on persistent TB. We designed a working drug screen assay to specifically target the mycobacterial sulfite reductase protein SirA. To this end we cloned Ito <i>E. coli </i><span style="font-style: normal;">the sulfite reduction pathway</span> of <i>M. smegmatis</i>, a non-pathogenic mycobacterial relative of <i>M. Tuberculosis</i>. Our model overcomes the problem of long doubling time of <i>M. tuberculosis</i>. Specific inhibition of the sulfite reduction pathway is scored by comparing a drug screen of our <i>E. coli</i> construct <i>vs.</i> wild-type. Any drug candidates that have activity against both the wild-type <i>E. coli</i> and our construct are non-specific inhibitors of <i>E. coli</i> growth. However, any drug candidates that inhibit only the growth of our <i>E. coli </i>construct will be <span style="font-style: normal;">SirA</span><i> </i><span style="font-style: normal;">pathway specific.</span> <br />
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<div class="rightparagraph"><br />
<a href="https://static.igem.org/mediawiki/2013/4/4f/PS_Drug_Scheme.png" target="_blank"><img src="https://static.igem.org/mediawiki/2013/4/4f/PS_Drug_Scheme.png" width="535px"/></a><br />
<p><b>Figure 1: Overview of Targeted Drug Screen Design</b></p><br />
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<div id="Model"></div><br />
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<h2>Flux Balance Analysis of Sulfite Reduction Pathway</h2><br />
<div class="leftparagraph"><br />
<p>&nbsp;&nbsp;<br />
We used an <i>E. coli</i> model (iJR904) obtained from the <a href="http://bigg.ucsd.edu/bigg/main.pl">BiGG database</a> as a starting model to obtain wild-type growth rate (f = 0.9129 divisions/hour). We then deleted the reaction ‘SULR’ which encodes for the sulphite reduction pathway involving cysI and obtained a f= -8e-13=0 divisions/hour indicating that the sulphite reduction pathway is essential for growth. Finally we introduced two new reactions for sirA and fprA and a new species fdxA. We found that growth with the mycobacteria pathway reverts the growth phenotype back to wild-type levels (f = 0.9105 divisions/hour). We then wanted to expand our model to find new pathways that we could utilize for a targeted drug screen approach. We wrote a matlab script that finds all the essential reactions in <i>M. tuberculosis</i> and all the essential reactions in <i>E. coli</i>, and then tries to complement the essential reactions in the <i>E. coli</i> model with the essential reactions from <i>M. tuberculosis</i>. The model identified <a href="https://2013.igem.org/Team:Paris_Bettencourt/Project/Target/FBA">100 metabolic reactions</a> that we could target. Additionally, due to the modular nature of the model, it can be used to find target-able metabolic reactions in any SBML file. The Matlab scripts can be found <a href="https://2013.igem.org/File:TargetFBA.zip">here</a> and requires <a href="http://opencobra.sourceforge.net/openCOBRA/Welcome.html">Cobra Toolbox 2.0</a> to function. Please visit the FBA page for a detailed list of <a href="https://2013.igem.org/Team:Paris_Bettencourt/Project/Target/FBA">results</a>.<br />
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<div class="rightparagraph"><br />
<center><a href="https://static.igem.org/mediawiki/2013/7/76/PS_FBA.png" target="_blank"><img src="https://static.igem.org/mediawiki/2013/7/76/PS_FBA.png" width="267.5px"/></a></center><br />
<p><b>Figure 2: Biomass Flux through <i>E. coli</i> and mycoSIR E. coli</b><div style="font-size: 90%">Flux balance analysis was run using Cobra Toolbox 2.0 on <i>E. coli</i> sbml model iJR904 with and without SULR reaction. Additionally an <i>E. coli</i> sbml model was built with the SULR reaction replaced with a reaction representing the mycobacterial SirA reaction and FprA reaction, as well as ferredoxin FdxA as an additional species. The Biomass flux is restored to 99.75% of the wild-type level with the synthetic mycobacterial system.</div></p><br />
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<div id="Model"></div><br />
<h2>Structural Analysis of SirA</h2><br />
<div class="leftparagraph"><br />
<p>&nbsp;&nbsp;<br />
Superimposing the structures of <i>M.tuberculosis</i> SirA and <i>E.coli </i> CysI reveals high homology, in particular of the active sites. Both proteins have the same symmetry (psuedo 2 fold) indicative of a common evolutionary origin. Our analysis highlighted important conserved residues, involved in substrate binding to be Arg97, Arg130, Arg166, Lys207. These positively charged residues are conserved in the sulphite/nitrite reductase family. In addition, 4 Cys residues are conserved for iron-sulphur binding. </p><br />
<p>The most profound structural differences between the two enzymes are found in the ferredoxin binding site and SirA's most C terminal residues and several surface loop regions due to deletions or insertions. A stark difference is a covalent bond formed between Cys161 (thiolate) and Tyr69 (C carbon atom) found adjacent to the redox center (Cu ions) in SirA. The covalently bound residues act as a secondary cofactor in tyrosyl radical stabilization. </p> <br />
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<div class="rightparagraph"><br />
<center><a href="https://static.igem.org/mediawiki/2013/5/52/Purplecys_SirA-1.png" target="_blank"><img src="https://static.igem.org/mediawiki/2013/5/52/Purplecys_SirA-1.png" width="267.5px"/></a></center><br />
<p><b>Figure 3: The superimposed 3D protein structures of SirA and CysI.</b><div style="font-size: 90%"> 303 amino acids are involved in superimposition with an rsmd of 1.41Å. All domains and loops of CysI are coloured purple, whilst SirA is coloured according to structural similarity with CysI: Red indicates poor alignment whilst blue indicates good alignment.</div></p><br />
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<div id="Model"></div><br />
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<h2>Identification of potential drug target binding sites</h2><br />
<div class="leftparagraph"><br />
<p>&nbsp;&nbsp;<br />
Our structural analysis provided the basis for our drug target prediction. Using Chembl and swiss pdb, we have shown a predicted drug target site. Our calculation gives strong favour for a drug to be effective at this site. The calculation reflects the suitability of small molecules to the binding site under the Lipinski's Rule of 5.</p><br />
<p>The drug target is located at the interface of the three domains. This binding pocket exhibits a dense hydrophobic region. Our analysis targets 48 amino acids of SirA within 6Å of a modelled small drug molecule. Of these residues, only 6 amino acids are charged: His409, Asp453, Asp474, His500, Asp504 and Arg541.<br />
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<div class="rightparagraph"><br />
<center><a href="https://static.igem.org/mediawiki/2013/5/59/Drug_target_withoutAA.png" target="_blank"><img src="https://static.igem.org/mediawiki/2013/5/59/Drug_target_withoutAA.png" width="267.5px"/></a></center><br />
<p><b>Figure 4 Drug target locations in SirA </b><div style="font-size: 90%">A domain located in SirA, identified as a drug target through Chembl analysis.</div></p><br />
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<h2>Structure based pharmacophore modelling of mycobacterial Fpra</h2><br />
<div class="leftparagraph"><br />
<p>&nbsp;&nbsp;<br />
Using LigandScount 3.1, we searched over 8100 drug compounds from the BindingDB and Chembl databases for drugs targetting mycobacterial Fpra. Our search revealed Riboflavin (Vitamin B2) and Pyridoxine to be drug targets for Fpra. We used NADP interacting with the active site as the model of the pharmacore. Results showed pyridoxin to be a competitive inhibitor to NADP. Pyridoxin is a synthetic compound currently available as a prescribed drug. </p><br />
<p>Chembl analysis of Pyridoxine (vitamin B6) show that it's properties fulfill Lipinski's criteria of being an orally active drug in humans. These properties state that any small drug molecule must have: no more than 5 H bond donors, no more 10 H bond acceptors (N or O atoms), mol mass of less than 500 dalts and octanol-water partition coefficient log P of no greater than 5).</p> <br />
<p>We have shown the proposed properties of Pyridoxine's interaction with Fpra as a competitive inhibitor to NADP at Fpra's active site. The key amino acids at the active site are Ala205, GLN204 and Thr208. GLN204 and Ala205 act as hydrogen bond acceptors whilst Thr208 interacts with a H via van der waals forces. Pyridoxin is a smaller, more lipid soluble molecule than NADP, thus more fitting to Lipinski's criteria. </p> <br />
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<div class="rightparagraph"><br />
<center><a href="https://static.igem.org/mediawiki/2013/4/42/PB_Fnr_ribbons3.png" target="_blank"><img src="https://static.igem.org/mediawiki/2013/4/42/PB_Fnr_ribbons3.png" width="100%"/></a></center><br />
<p><b>Figure 5: </b><div style="font-size: 90%">Our 3D model shows the structure of FNR where negative residues are coloured in blue, positive residues in red and NAD in purple (ball and stick representation). The key amino acids at the active site are Glu211, Gly 366, Arg 110, Arg 199, Arg 200 and Asn155. Glu211 acts as a hydrogen acceptor whilst the latter four residues act as hydrogen donors.</div></p><br />
<center><a href="https://static.igem.org/mediawiki/2013/0/0a/PB_picture16.15.png" target="_blank"><img src="https://static.igem.org/mediawiki/2013/0/0a/PB_picture16.15.png" width="100%"/></a></center><br />
<p><b>Figure 6:</b></b><div style="font-size: 90%"> The interaction of Pyridoxine to its active site residues.</div> </p><br />
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<div id="Design"></div><br />
<h2>Synthetic Mycobacteria Pathway</h2><br />
<div class="leftparagraph"><br />
<p> &nbsp;&nbsp;<br />
We designed a synthetic <i>M.smegmatis-</i> derived sulfite reduction pathway containing sirA - the sulfite reductase, and two supporting genes that are required for its function in <i>E.coli</i>: fdxA and fprA. FdxA is a mycobacterial Ferredoxin cofactor which is oxidised by SirA during the sulfite reduction reaction and FprA is a Ferredoxin-NADPH reductase use replenish the reduced Fdx pool. The genes' sequences were taken from previous work describing their expression <a href="#Reference">(Pinto <i>et al</i> 2007)</a> in <i>E.coli</i> for purification and in vitro characterization; we removed restriction sites and codon optimized for expression in <i>E. coli</i>. The genes were then cloned into two Duet expression vectors, one containing sirA and one containing the supporting genesand were transformed into our knock-out mutant strains of <i>E. coli</i>. Data on Growth curves can be found <a href="https://2013.igem.org/Team:Paris_Bettencourt/Notebook#target_Monday_30th_September.html">here</a>. <br />
</p><br />
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<div class="rightparagraph"><br />
<a href="https://static.igem.org/mediawiki/2013/8/84/TB_drug_FinalSmeg.png" target="_blank"><img src="https://static.igem.org/mediawiki/2013/8/84/TB_drug_FinalSmeg.png" width="535px"/></a><br />
<p><b>Figure 7: Growth curves of <i>E. coli</i> mycoSIR</b> <div style="font-size: 90%">BL21 (DE3) ΔcysI containing the MycoSIR pathway (MycoSIR <i>E. coli</i>) were grown in liquid minimal media containing Various concentration of IPTG. (A) Replicates of each strain were measured for absorbance in a spectrophotometer every 10 minutes for 14 hours. Growth was observed for the WT BL21 <i>E. coli</i>, (blue), and the MycoSIR <i>E. coli</i> (red). No growth was detected for uninduced MycoSIR <i>E. coli</i> (purple) or for the BL21 (DE3) ΔcysI that did not contain the synthetic pathway (Orange) . (B) Mean Final ODs of all replicates, measured after 14 hours of growth. Growth was detected in zmSIR <i>E. coli</i> and WT BL21 but not in uninduced zmSIR strain.</div></p> <br />
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<h2>Creation of Knock out Mutants</h2><br />
<div class="leftparagraph"><br />
<p>&nbsp;&nbsp;<br />
We prepared two strains of <i>E. coli</i> which have the sulfite reduction pathway deleted: BL21 (DE3) <i>ΔCysI Δfpr ΔydbK</i> and BL21 (AI) <i>ΔCysI</i>. CysI is responsible for sulfite reduction in <i>E. coli</i>, while <i>fpr and ydbK</i> are two non-essential genes that consume ferredoxin. These two genes are deleted, as sulfite reduction in mycobacteria is ferredoxin dependent in comparison to<i> E. coli</i> in which it is NADPH dependant. These genes were also removed to ensure that they do not interfere with our system. <br />
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<div class="rightparagraph"><br />
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<h2>Synthetic Corn Pathway</h2><br />
<div class="leftparagraph"><br />
<p>&nbsp;&nbsp;<br />
Additionally we prototyped the system with a reconstruction of a sulphite reduction pathway previously designed and published by the silver group <a href="#Reference">(2011 Barstow et al)</a>. In place of CysI, a corn (Zea mays) derived sulfite reductase (zmSIR) was used. Two additional genes were included: Spinach ferredoxin (soFD), and corn derived ferredoxin NADP+ reductase (zmFNR). These genes, respectively, are required for production of the ferredoxin cofactor and the NADP+ ferredoxin reductase and are required for sulfite reductase (zmSIR) to function within <i>E. coli</i>.<br />
</p><br />
</div><br />
<div class="rightparagraph"><br />
<a href="https://static.igem.org/mediawiki/2013/a/a2/PB_final_Corn.png" target="_blank"><img src="https://static.igem.org/mediawiki/2013/a/a2/PB_final_Corn.png" width="535px"/></a><br />
<p><b>Figure 8:</b> Growth curves of <i>E. coli</i> maizeSIR<div style="font-size: 90%"> BL21 (DE3) ΔcysI containing the MaizeSIR pathway (MaizeSIR <i>E. coli</i>) were grown in liquid minimal media containing Various concentration of IPTG. (A) Replicates of each strain were measured for absorbance in a spectrophotometer every 10 minutes for 14 hours. Growth was observed for the WT BL21 <i>E. coli</i>, (blue), and the MaizeSIR <i>E. coli</i> (red). No growth was detected for uninduced MaizeSIR <i>E. coli</i> (purple) or for the BL21 (DE3) ΔcysI that did not contain the synthetic pathway (Orange) . (B) Mean Final ODs of all replicates, measured after 14 hours of growth. Growth was detected in zmSIR <i>E. coli</i> and WT BL21 but not in uninduced zmSIR strain.</div></p> <br />
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<div id="Results"></div><br />
<h2>Results</h2><br />
<div class="leftparagraph"><br />
<p>&nbsp;&nbsp;<br />
Upon successful cloning of the three genes into our <i>E. coli</i> deletion strain, we continued to confirm that all three genes are required for growth on minimal media. Our two synthetic pathways were found to rescue growth on a sulfurless amino acid supplemented minimal media. We hope that this technique of using synthetic biology to overcome problems faced in naturally occurring systems will be both a large boon to the pursuit of finding novel drug candidates in <i>M. tuberculosis</i> and more broadly as this technique can be used for high-throughput screening of any pathway that can be constructed to be essential for growth in <i>E. coli</i>.<br />
</p><br />
</br><br />
<p><b>Figure 9:</b> Growth of zmSIR <i>E. coli</i> on minimal media. <div style="font-size: 90%">BL21 (DE3) ΔcysI cells transformed with 1, 2 and 3 genes of the 3-gene zmSIR synthetic pathway were grown for 24 hours on minimal media supplemented with 25 uM IPTG (see methods), along with a WT BL21 (DE3) serving as a negative control, and an untransformed BL21 (DE3) ΔcysI, as negative control. Rescue of growth required all genes of the synthetic pathway (SIR, FNR and FD). </div></p><br />
</div><br />
<div class="rightparagraph"><br />
<a href="https://static.igem.org/mediawiki/2013/8/85/PS_D_Figure1_plate.png" target="_blank"><img src="https://static.igem.org/mediawiki/2013/8/85/PS_D_Figure1_plate.png" width="535px"/></a><br />
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<h2>Z-score</h2><br />
<div class="leftparagraph"><br />
<p>&nbsp;&nbsp;<br />
The Z-score is a statistical measurement aimed at assessing the "hit effect" in a drug screen high throughput screening. It is a commonly used measurement that shows how well did the drug effect the growth of the assay strain and how significant is the decrease in growth.</p><br />
<p><br />
To calculate the Z-score we used our experimental <i>E. coli</i> strain BL21 (AI) ΔcysI that carries all three genes of the synthetic pathway (sirA, fprA, fdxA). We grew it in the M9 minimal media supplemented with amino acid sulfur dropout powder, in a 96 well plate. Four of the wells were "spiked" with antibiotics (Amp, Gent, Kan, and Spect). </p><br />
</div><br />
<div class="rightparagraph"><br />
<p>&nbsp;&nbsp;<br />
This served as a simulation of the drug screen without the actual drug library. Only the drug screen controls are used: growth in M9 as a negative control (no drugs) and growth in M9 + antibiotics as a positive control (a sure hit). We then compared the distribution of the growth (OD) in the negative control with the distribution of growth (OD) in the positive control. The Z-score shows the distance of the negative control mean from the positive control mean in negative control standard deviation units.</p><br />
<p><b>Our Z-score is: -10.2.</b></p><br />
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<br />
<h2>Z-factor</h2><br />
<div class="leftparagraph"><br />
<p>&nbsp;&nbsp;<br />
Z-factor is a measurement complementary to the Z-score. It measures the assay's quality based on the same data extracted from the same experiment made for the Z-score. This calculation gives an estimation of how far the negative controls are from the positive controls. It is a comparison of the two distributions which assumes that both distributions are normal and calculate how far 99% of the data points of each distribution are from each other.</p><br />
<br />
</div><br />
<div class="rightparagraph"><br />
<p>&nbsp;&nbsp;<br />
Z-factor is given on a scale from 0 to 1. Scores between 0.5 and 1 show that the assay is good and will enable testing in High throughput screens.</p><br />
<p><b>Our Z-factor score is 0.58.</b></p><br />
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<div style="clear: both;"></div><br />
<br />
<h2> MycoSir growth assays reveal the potential anti TB activity of Pyridoxine </h2><br />
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<div class="leftparagraph"><br />
<p>&nbsp;&nbsp;</p><br />
</div><br />
<div class="rightparagraph"><br />
<p>&nbsp;&nbsp;</p><br />
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<div style="clear: both;"></div><br />
<br />
<div class="leftparagraph"> <br />
<center><a href="https://static.igem.org/mediawiki/2013/d/d5/PB_RiboflavinData.png"><img width="80%" src="https://static.igem.org/mediawiki/2013/d/d5/PB_RiboflavinData.png"/></a></center><br />
<p><b>Figure 10:</b>Riboflavin has no effect on the growth of WT or synthetic MycoSIR <i>E. coli.</i><div style="font-size: 90%"></div> The indicated quantities of riboflavin were dissolved in water and added to cultures of WT or MycoSIR E. coli in 4 biological replicates. No significant growth effects were observed. </p><br />
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</div><br />
<div class="rightparagraph"><br />
<center><a href="https://static.igem.org/mediawiki/2013/8/86/PyridoxineData.png"><img width="80%" src="https://static.igem.org/mediawiki/2013/8/86/PyridoxineData.png"></a></center><br />
<p><b>Figure 11:</b> MycoSIR E. coli growth assays reveal a potential anti-TB acitivity of pyridoxine at high doses. <div style="font-size: 90%"> The indicated quantities of pyridoxine were dissolved in water and added to cultures of WT or MycoSIR E. coli in 4 biological replicates. Both strains were grown in defined minimal media, where MycoSIR E. coli require our synthetic pathway for growth. Low pyridoxine doses had no detectable effects. However, a very high dose of pyridoxine (10 mg/mL) substantially inhibited the growth of MycoSIR E. coli yet showed no effect on WT growth. This suggests pyridoxine specifically inhibits the activity of the Mycobacterial SirA pathway. Derivativization or other methods could be used to further enhance the affinity and specificity of this compound.</div> </p><br />
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<div id="Reference"></div><br />
<h2>Literature</h2><br />
<div class="leftparagraph"><br />
<ul><br />
<li>Global Alliance for TB Drug Development, Tuberculosis. Scientific blueprint for tuberculosis drug development, Tuberculosis (Edinb) 81 Suppl 1, 1–52 (2001).</li><br />
<br />
<li>World Health Organization, Global Tuberculosis Report 2012 (2012).</li><br />
<br />
<li>K. Raman, K. Yeturu, N. Chandra, targetTB: A target identification pipeline for Mycobacterium tuberculosis through an interactome, reactome and genome-scale structural analysis, BMC Syst Biol 2, 109 (2008).</li><br />
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<li>R. Pinto, J. S. Harrison, T. Hsu, W. R. Jacobs, T. S. Leyh, Sulfite Reduction in Mycobacteria, Journal of Bacteriology 189, 6714–6722 (2007).</li><br />
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<li>B. Barstow C. M. Agapakis, P. M. Boyle, G. Grandl, P. A. Silver, E. H. Wintermute, A synthetic system links FeFe-hydrogenases to essential E. coli sulfur metabolism, J Biol Eng 5, 7 (2011).</li><br />
</ul><br />
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</div><br />
<div class="rightparagraph"><br />
<ul><br />
<li>Schellenberger J, Que R, Fleming RMT, Thiele I, Orth JD, Feist AM, Zielinski DC, Bordbar A, Lewis NE, Rahmanian S, Kang J, Hyduke DR, Palsson BØ. 2011 Quantitative prediction of cellular metabolism with constraint-based models: the COBRA Toolbox v2.0. Nature Protocols 6:1290-1307.</li><br />
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<li>Schellenberger, J., Park, J. O., Conrad, T. C., and Palsson, B. Ø., BiGG: a Biochemical Genetic and Genomic knowledgebase of large scale metabolic reconstructions, BMC Bioinformatics, 11:213, (2010).</li><br />
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<li>S. G. Franzblau et al., Comprehensive analysis of methods used for the evaluation of compounds against Mycobacterium tuberculosis, Tuberculosis 92, 453–488 (2012).</li><br />
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<li>D. J. Payne, M. N. Gwynn, D. J. Holmes, D. L. Pompliano, Drugs for bad bugs: confronting the challenges of antibacterial discovery, Nat Rev Drug Discov 6, 29–40 (2006).</li><br />
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<li>M. Nakayama, T. Akashi, T. Hase, Plant sulfite reductase: molecular structure, catalytic function and interaction with ferredoxin, J. Inorg. Biochem. 82, 27–32 (2000).</li><br />
</ul><br />
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<br />
<h2>Attributions</h2><br />
<div class="leftparagraph"><br />
<ul><br />
<li>Strains NEBTurbo, BL21 (DE3) KO20, BL21 AI were provided by INSERM U1001.</li><br />
<li>Plasmids pET Duet, pACYC Duet, pACYC zmSIR, pACYC soFD zmSIR, pCDF FNR were provided by INSERM U1001.</li><br />
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<li>Genes msSirA, msFprA, msFdxA were synthesized by IDT.</li><br />
<li>Project was designed by Idonnya Aghoghogbe, Yonatan Zegman, Matthew Deyell and Edwin Wintermute. All experiments and modelling were performed by Idonnya Aghoghogbe, Yonatan Zegman, Matthew Deyell. </li><br />
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{{:Team:Paris_Bettencourt/footer}}</div>Idonnyahttp://2013.igem.org/Team:Paris_Bettencourt/CollaborationTeam:Paris Bettencourt/Collaboration2013-10-29T03:40:04Z<p>Idonnya: </p>
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<div class="hlink"><br />
<h2>Skip to Calgary</h2><br />
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<a href="#BGU"><br />
<div class="hlink"><br />
<h2>Skip to BGU </h2><br />
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<a href="#Braunschweig"><br />
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<h2>Skip to Braunschweig</h2><br />
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<p> In Science, it is all about collaborating. Working together makes better science and is fun! Throughout our iGEM summer, we have been in contact with several teams and successfully collaborated with 3 teams from 3 different continents! As part of our <a href="https://2013.igem.org/Team:Paris_Bettencourt/Project/Detect" target="_blank"> Biosensor project</a>, we collaborated with the <a href="https://2013.igem.org/Team:Calgary" target="_blank"> Calgary team, Canada</a>, and built a biosensor iGEM database – <a href="http://www.sensigem.org"> sensiGEM</a>. With the <a href="https://2013.igem.org/Team:BGU_Israel"> BGU Israel</a> team, we collaborated in an experimental aspect. They sent us parts of them to clone and in return they did a western blot for us, which supports the work of the <a href="https://2013.igem.org/Team:Paris_Bettencourt/Project/Infiltrate"> Infiltrate subproject</a>. The <a href="https://2013.igem.org/Team:Braunschweig"> Braunschweig Team</a> from Germany was supported from us with literature they needed to continue working on their project. Below you can find a description as well as the outcome of our collaborations! As we have collaborated successfully with our partners we further provide some tips for how we think iGEM Teams can successfully collaborate. </p><br />
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<div id="Calgary"></div><br />
<h2>Collaboration with the Calgary iGEM Team from Canada</h2><br />
<div class="leftparagraph"><br />
<p> &nbsp;&nbsp; The Paris-Bettencourt-Calgary iGEM collaboration started last June when a few members from each team met at the SB6.0 synbio conference in London. After a few beers and lab stories, we learned that despite coming from the opposite sides of the globe, we were using synthetic biology to build biosensors to sense DNA. While our systems were targeted solve different problems, we were struck by a number of commonalities between these projects (Figure 1). As we shared our projects, we recalled how there was a lack of DNA biosensor parts in the Parts Registry. Moreover, we complained about the lack of organization of biosensors in the registry. The veteran iGEMers on each team mentioned that biosensors had consistently finished as grand prize winners in previous years of iGEM. <br />
</p><br />
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<div class="rightparagraph"><br />
<p>We were curious how biosensors have evolved since the beginning of iGEM and how our projects fit into the context of the iGEM Parts Registry.</p><br />
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<img src="https://static.igem.org/mediawiki/2013/3/31/PB_diagrammcomparaisionpbcalgary.png" width="100%"><br><br />
<b>Figure 1.</b> The Calgary and Paris Bettencourt biosensors both sense DNA, albeit with some differences in how they function mechanistically.<br />
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<h3>SensiGEM - A biosensor database</h3><br />
<div class="leftparagraph"><br />
<p><br />
&nbsp;&nbsp; We decided to collaborate to answer these questions. Since our initial meeting in London, members of each team have conferenced weekly on Skype. After accustoming ourselves to the eight hour time difference, we developed SensiGEM, a collaborative database in which we catalogued all the biosensors in the history of iGEM.<br />
</p><br />
<p><br />
Before studying the past Wikis, we realized that we had different definitions of biosensors. We asked each other a fundamental question: What is a biosensor? We developed the following definition: A biosensor is an engineered system that relies on biological systems or components to detect and report a condition. The condition(s) detected and reported could encompass an environmental, biological, chemical or synthetic aspect or compound in the sensor’s environment or surroundings.<br />
Once agreeing on the nature of biosensors, we split up the Wikis from 2007 onward between Calgary and Paris-Bettencourt. We analyzed 936 project Wikis from 2007 to 2013 by hand, incorporating the projects which matched our biosensor definition into the collaborative SensiGEM database. We included 229 projects on </p><br />
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<div class="rightparagraph"><br />
<p> the database, some of which were biosensors as per the definition, as well as other projects containing biosensor elements that aligned with our definition.</p><br />
<p>We designed this database with future iGEM teams in mind, with tools for efficient navigation biosensors according to inputs, outputs, and their intended application. We made both SensiGEM’s source code and underlying data available under the permissive MIT license. This means that other teams can either collaborate with us on our version of the database or host their own independent copies. We foresee SensiGEM as a resource where future iGEM teams can showcase their biosensors.<br><br />
<br><br />
<a class="sensiGEM" href="http://www.sensigem.org/"><br />
<center><img src="https://static.igem.org/mediawiki/2013/8/8e/CollaborationParisCalgary.png" width="40%"></center><br />
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<h3>Lessons from SensiGEM</h3><br />
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<p>&nbsp;&nbsp; We conducted some preliminary analysis of the database in SensiGEM to see how our projects stand in the current iGEM biosensor landscape as well as to get an overview of what types of biosensors have been developed for iGEM.<br><br />
In Figure 2, we can see the portion of biosensors of all iGEM projects since 2007. There is a clear linear increase in number of iGEM projects, but the number of biosensors per year varies and doesn’t follow a trend.<br><br />
How successful Biosensor were in the previous years (Advance to Championship, Awards, Finalists) can be seen in Figure 3. Something that also interested us was to see how many biosensors are in the Track Health and Medicine as we developed a biosensor for that Category (Figure 4). Other facts we wanted get from the database are, how many biosensors already targeted DNA (Figure 5) and what is the distribution of other inputs, sensed by previous biosensors (Figure 6).</p><br />
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<img src="https://static.igem.org/mediawiki/2013/3/3c/PB_collabofigure5.png" width="500"><br><br />
<b>Figure 5.</b> Number of iGEM biosensor projects in comparison to how many of these targeted DNA like the Calgary team and we did.<br><br />
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<img src="https://static.igem.org/mediawiki/2013/1/16/PB_collaboration_Figure_6_cake.png" width="500"><br><br />
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<b>Figure 6.</b> Overview of the different types of inputs for the biosensors in iGEM and how many teams used those inputs since 2007.<br><br />
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<br><br />
<img src="https://static.igem.org/mediawiki/2013/8/8c/PB_collabofigure2.png" width="500"><br><br />
<b>Figure 2.</b> Number of iGEM projects since 2009 in comparison to number of Biosensor iGEM projects since 2007.<br><br />
<br><br />
<img src="https://static.igem.org/mediawiki/2013/4/4c/PB_collabofigure3.png" width="500"><br><br />
<b>Figure 3.</b> Success of Biosensor iGEM projects since 2007. Listed are the numbers of teams that advanced to the Championship (since 2009), that won awards or were finalists (since 2007).<br><br />
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<img src="https://static.igem.org/mediawiki/2013/d/d8/PB_collabofigure4.png" width="500"><br><br />
<b>Figure 4.</b> Number of iGEM projects since 2007 in the Track Helath and Medicine and how many of these are/were biosensors<br><br />
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<h3>Looking toward finals</h3> <br />
<div class="leftparagraph"><br />
<p> &nbsp;&nbsp; Given the similarities between each of our systems in overall function, we have begun development of BioBricks to apply each system to the other team's problem. By testing each system on a different problem, we intend to show how each system can be deployed as a modular, platform technology.</p><br />
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<div class="rightparagraph"><br />
<p>We also intend to further improve our database to more easily get information of the actual sensing system (sensitive/inducible promoter, other targeting methods like CRISPRs, TALEs,…).</p><br />
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<div id="BGU"></div><br />
<h2>Collaboration with the BGU iGEM Team from Israel</h2><br />
<div class="leftparagraph"><br />
<p>A critical part of BGU's project requires a recombination of a linear DNA cassette into the bacterial chromosome. They sent a cassette required for the recombination as well as the helper plasmid required for it to function. They sent us their plasmid pUC57amp-cI which is responsible for the repression of the holin and lysozyme genes in their kill switch. As the repression of these genes is essential to prevent the kill switch from being lethal prematurely, we decided to characterize the promoter units produced by the lac/ara-1 promoter that the cI is controlled by.</p></div> <div class="rightparagraph"><p>The first thing we needed to do was to biobrick the promoter from pUC57amp-cI. We PCR'd biobrick restriction sites onto the lac/ara-1 promoter along with the RBS. We then cloned the lac/ara-1 promoter into pSB1C3 backbone. We extracted the promoter from pSB1C3 with EcoRI and SpeI cut sites, and a GFP reporter E0240 with XbaI and PstI and the backbone was prepared with EcoRI and PstI as per the protocol for characterizing promoter activity on the biobrick website. At time of writing we are currently cloning the lac/ara-1 GFP construct for characterization.</p></div><br />
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<div id="Braunschweig"></div><br />
<h2>Collaboration with the Braunschweig iGEM Team from Germany</h2> <br />
<div class="leftparagraph"><br />
<p> Jonas Zantow an advisor of the Braunschweig iGEM Team from Germany visited us in Paris. We we showed him our lab and we gave him a quick overview of our projects and what we achieved so far. Also Jonas explained us what the Braunschweig 2013 iGEM Team <br><br />
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<div class="rightparagraph"><br />
<p> is working on and we could help him out with some Literature from our Bibliography we started during our Brainstorming sessions earlier the year. His visit ended - how can it be different, when we have German visitors - over a glass of beer! </p><br />
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<div id="Tips"></div><br />
<h2>Tips for how you can successfully collaborate</h2> <br />
<div class="leftparagraph"><br />
<p> Important for the success of our cooperation was to regularly see each other on Skype. It is way easier to talk in person than to communicate by mail as misunderstandings and questions could be solved directly. We used Asana.com to set up tasks for each team that completed until the next week. Setting up those weekly tasks was a good idea as we were not overwhelmed by a lot of work but having those small tasks we could process them in time. Also the splitting up of work, as we did for example for the Wikis, was very helpful and part of the success to reach our goal in time.<br><br />
</p><br />
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<div class="rightparagraph"><br />
<p>For future collaborations, setting up weekly tasks and skyping every week worked well for us! Also important is that both teams have the same idea of the aim of the collaboration as well as how to achieve it. With the Calgary Team 2013 we found a great partner that fit to what we imagined!</p><br />
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{{:Team:Paris_Bettencourt/footer}}</div>Idonnyahttp://2013.igem.org/Team:Paris_Bettencourt/PartsTeam:Paris Bettencourt/Parts2013-10-29T03:36:02Z<p>Idonnya: </p>
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<p><b> Here, you can find parts as well as backbones that we submitted to the parts registry. With the new submitted BioBricks highly valued constructs are now added to the registry as several sRNAs. <br />
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<div class="rightparagraph"><br />
<p><b>We also improved the standard BioBrick vector, which has now compatible origins of replication and resistances. Next to that you can find our suggestion for a novel assembly standard.<br />
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<a href="https://2013.igem.org/Team:Paris_Bettencourt/BioBricks" title="BioBricks"><br />
<h2>BioBricks</h2><br />
</a><br />
<p><a href="https://2013.igem.org/Team:Paris_Bettencourt/BioBricks">We submitted eleven new biobricks </a> to the parts registry. Each sub-project submitted parts so you can find e.g. the gRNA anti KAN from Detect, the SirA gene of <i>M. Smegmatis</i> of the Target team, the TDMH gene from Infiltrate and sRNA anti Cm from the Sabotage team. <br />
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<h2>BackBones</h2><br />
</a><br />
<p><a href="https://2013.igem.org/wiki/index.php?title=Team:Paris_Bettencourt/Backbones">We upgraded</a> the standard biobrick shipping vector pSB1C3 with a new set of 4 ori’s and antibiotic resistance cassettes so that they can easily be co-expressed.The system of 4 backbones all have compatible origins and resistance cassettes that allows for up to 4 back bones to be expressed in a single host bacteria. <br />
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<h2>Assembly Standard</h2><br />
</a><br />
<p><a href="https://2013.igem.org/Team:Paris_Bettencourt/Assembly_Standard">We offer a new assembly standard</a>. It enables keeping the BioBrick standard while providing the needed tools to perform assembly of several parts in one step. BBG is a fusion of the BioBrick standard cloning and Gibson isothermal assembly. <br />
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{{:Team:Paris_Bettencourt/footer}}</div>Idonnyahttp://2013.igem.org/Team:Paris_Bettencourt/ResultsTeam:Paris Bettencourt/Results2013-10-29T02:55:28Z<p>Idonnya: </p>
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<h2>Detect</h2><br />
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<h2>Target</h2><br />
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<h2>Infiltrate</h2><br />
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<h2>Sabotage</h2><br />
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<h2><a href="https://2013.igem.org/Team:Paris_Bettencourt/Project/Detect">Detect</a></h2><br />
<div class="overbox"><br />
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<h2>Background</h2><br />
<p>CRISPR/Cas systems generate site-specific double strand breaks and have recently been used for genome editing. </p><br />
</div><br />
<div class="aims"><br />
<h2>Aims</h2><br />
<p>Building a genotype sensor based on CRISPR/Cas that reports existance of an antibiotic resistance gene.</p><br />
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<div class="subbox2"><br />
<div class="results"><br />
<h2>Results</h2><br />
<ul><br />
<li>Successfully cloned gRNA anti-KAN, crRNA anti-KAN, tracrRNA-Cas9 and pRecA-LacZ into Biobrick backbones and therefore generated four new BioBricks. </li><br />
<li> Confirmation of site-specific binding and DNA double-strand breaks generated by the gRNA-Cas9 complex in the kanamycin resistance casette. </li><br />
<li>Testing the new assembly standard for our cloning.</li><br />
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<a href="http://parts.igem.org/Part:BBa_K1137014" target="_blank"><img width="24%" style="margin-top:15px" src="https://static.igem.org/mediawiki/2013/a/ab/Bb_detect_cas9.png"/></a><br />
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<a href="http://parts.igem.org/Part:BBa_K1137013" target="_blank"><img width="24%" style="margin-top:15px" src="https://static.igem.org/mediawiki/2013/9/97/Bb_detect_crRNAKan.png"/></a><br />
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<a href="http://parts.igem.org/Part:BBa_K1137012" target="_blank"><img width="24%" style="margin-top:15px" src="https://static.igem.org/mediawiki/2013/7/7b/Bb_detect_gRNAkan.png"/></a><br />
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<a href="http://parts.igem.org/Part:BBa_K1137015" target="_blank"><img width="24%" style="margin-top:15px" src="https://static.igem.org/mediawiki/2013/0/05/Bb_detect_LacZ.png"/></a><br />
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<br><br />
<br />
<p style="margin-top:-60px;font-size:13px"><b>CRISPR anti-Kan plasmids target specifically kanamycin resistant E. coli. </b>We introduced our CRISPR-based DNA cleavage system to two strains of E. coli : one WT (blue bars) and one carrying a genomically integrated kanamycin resistance casette (KanR, blue bars). The strains were co-transformed with two plasmids. One with the Cas9 construct, the other with the anti-Kanamycin gRNA. WT E.coli could be efficiently transformed with one or both plasmids, as determined by selective plating. However, KanR E. coli could not be efficiently transformed with both. We attribute this to Cas9-induced cleavage of the chromosome specifically at the KanR casette, with about 99% efficiency.<br />
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<h2><a href="https://2013.igem.org/Team:Paris_Bettencourt/Project/Target">Target</a></h2><br />
<div class="overbox"><br />
<div class="subbox1"><br />
<div class="bkgr"><br />
<h2>Background</h2><br />
<p>SirA is an essential gene in latent tuberculosis infections. </p><br />
</div><br />
<div class="aims"><br />
<h2>Aims</h2><br />
<p>To perform a drug screen targeted at the sirA gene from mycobacteria.</p><br />
</div><br />
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<div class="subbox2"><br />
<div class="results"><br />
<h2>Results</h2><br />
<ul><br />
<li>Produced an E. coli strain which relies upon mycobacterial sirA, fprA and fdxA genes to survive in M9 minimal media.</li><br />
<li>Demonstrated that E. coli can survive with mycobacterial sulfite reduction pathway with Flux Balance Analysis.</li><br />
<li>Located drug target sites on sirA as well as identified high structural similarity between cysI and sirA through structural analysis.</li><br />
</ul><br />
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<a href="http://parts.igem.org/Part:BBa_K1137000" target="_blank"><img height="80%" style="margin-top:15px;margin-left:30px" src="https://static.igem.org/mediawiki/2013/7/79/Bb_target_sirA.png"/></a><br />
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<a href="http://parts.igem.org/Part:BBa_K1137001" target="_blank"><img height="80%" style="margin-top:15px" src="https://static.igem.org/mediawiki/2013/c/cb/Bb_target_fprA.png"/></a><br />
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<a href="http://parts.igem.org/Part:BBa_K1137002" target="_blank"><img height="80%" style="margin-top:15px" src="https://static.igem.org/mediawiki/2013/4/40/Bb_target_fdxA.png"/></a><br />
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<div class="mainfig"><br />
<center> <img width="100%" src="https://static.igem.org/mediawiki/2013/3/31/PB_fig_target1.png"/></center><br />
<p style="font-size:13px"> <b>MycoSIR E. coli depend on our synthetic pathway for growth.</b> E. coli strain BL21(DE3) was deleted for cysI and transformed with the three genes of the mycoSIR pathway expressed from IPTG-inducible T7 promoters (red). Wild-type (blue), uninduced (purple) and pathway-minus (gold) strains were used as controls. Both time course growth curves (A) and final ODs (B) reveal that the complete, induced pathway is required for growth <br />
</p><br />
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<div id="Infiltrate"></id><br />
<h2><a href="https://2013.igem.org/Team:Paris_Bettencourt/Project/Infiltrate">Infiltrate</a></h2><br />
<div class="overbox"><br />
<div class="subbox1"><br />
<div class="bkgr"><br />
<h2>Background</h2><br />
<p>Latent tuberculosis persists inside macrophages of the lungs, where it is partially protected from both the host immune system and conventional antibiotics. </p><br />
</div><br />
<div class="aims"><br />
<h2>Aims</h2><br />
<p>To create an <i>E. coli</i> strain capable of entering the macrophage cytosol and delivering a lytic enzyme to kill mycobacteria.</p><br />
</div><br />
</div><br />
<div class="subbox2"><br />
<div class="results"><br />
<h2>Results</h2><br />
<ul><br />
<li>We expressed the enzyme Trehalose Dimycolate Hydrolase (TDMH) in <i>E.coli</i> and showed that it is highly toxic to mycobacteria in culture.</li><br />
<li>We expressed the lysteriolyin O (LLO) gene in <i>E. coli</i> and showed that it is capable of entering the macrophage cytosol.</li><br />
<li>We co-infected macrophages with both mycobacteria and our engineered <i>E. coli</i> to characterize the resulting phagocytosis and killing.</li><br />
</ul><br />
</div><br />
<div class="biocriks"><br />
<br />
<a href="http://parts.igem.org/Part:BBa_K1137008" target="_blank"><img height="80%" style="margin-top:15px; margin-left:150px" src="https://static.igem.org/mediawiki/2013/a/a0/Bb_infiltrate_tdmh.png"/></a><br />
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</div><br />
</div><br />
<div class="mainfig"><br />
<center><br> <img width="95%" src="https://static.igem.org/mediawiki/2013/b/bb/PB_fig_Infil.png"/></center><br />
<p style="font-size:13px"> <b>TDMH expression kills mycobacteria in culture.</b> We mixed E. coli and WT M.smegmatis in LB media. Plating assays were used to count specifically M. smegmatis after the indicated times. When TDMH-expression was fully induced, more than 98% of mycobacteria were killed after 6 hours (red line). Populations of mycobacteria alone (black line) and mycobacteria mixed with uninduced E. coli (blue line) were stable.<br />
</p><br />
</div><br />
</div><br />
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<br />
<div id="Sabotage"></id><br />
<h2><a href="https://2013.igem.org/Team:Paris_Bettencourt/Project/Sabotage">Sabotage</a></h2><br />
<div class="overbox"><br />
<div class="subbox1"><br />
<div class="bkgr"><br />
<h2>Background</h2><br />
<p>One of the main concerns about tuberculosis today is the emergence of antibiotic resistant strains.</p><br />
</div><br />
<div class="aims"><br />
<h2>Aims</h2><br />
<p>Our objective is to make an antibiotic-resistant bacterial population sensitive again to the selfsame antibiotics.</p><br />
</div><br />
</div><br />
<div class="subbox2"><br />
<div class="results"><br />
<h2>Results</h2><br />
<ul><br />
<li>Construction and characterization of phagemids coding for small RNA targeting antibiotic resistance proteins.</li><br />
<li>Showed theoretically burden of a device is critical for the maintenance of a genetic element in a population.</li><br />
<li>Successful conversion of antibiotic resistant population of E. coli to a sensitive state (on two different antibiotic resistances).</li><br />
<br />
</ul><br />
</div><br />
<div class="biocriks"><br />
<br />
<a href="http://parts.igem.org/Part:BBa_K1137009" target="_blank"><img height="80%" style="margin-top:15px;margin-left:30px" src="https://static.igem.org/mediawiki/2013/3/37/Bb_sabotage_aKAN.png"/></a><br />
<br />
<a href="http://parts.igem.org/Part:BBa_K1137010" target="_blank"><img height="80%" style="margin-top:15px" src="https://static.igem.org/mediawiki/2013/6/66/Bb_sabotage_aCm.png"/></a><br />
<br />
<a href="http://parts.igem.org/Part:BBa_K1137011" target="_blank"><img height="80%" style="margin-top:15px" src="https://static.igem.org/mediawiki/2013/7/72/Bb_sabotage_aLac.png"/></a><br />
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</div><br />
</div><br />
<div class="mainfig"><br />
<center><br> <img width="70%" src="https://static.igem.org/mediawiki/2013/b/b2/Newfiguresabotagepresentation.png"/></center><br />
<p style="font-size:13px"><b>Our synthetic phage conveys antibiotic-sensitivity to an antbiotic-resistant population. </b>The anti-Chloramphenicol phage system effectively killed 99.1% of the Chloramphenicol resistant population and the anti- Kanamycine phage effictevely killed 99,5% of the Kanamycine resistant population.<br />
</p><br />
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<h2><a href="">Modelling</a></h2><br />
<div class="overbox" style=""><br />
<div class="projtile" style=""><br />
<h2><a href="https://2013.igem.org/Team:Paris_Bettencourt/Project/Sabotage">Population Dynamics Model</a></h2><br />
<p>This model investigates the propagation of horizontal gene-transfer via phagemid/helper system. We study the effect of the burden of a desired device on the maintenance of the system in time.</p><br />
</br><br />
<center><a href="https://static.igem.org/mediawiki/2013/3/38/PB_Model_diagram3.png" target="_blank"><img src="https://static.igem.org/mediawiki/2013/3/38/PB_Model_diagram3.png" width="80%"/></a></center><br />
</br><br />
<p style="font-size:13px">Left: scheme representing the regular non-lytic M13 bacteriophage horizontal spread. Right: scheme representing the main processes of the phagemid/helper system.</p> <br />
</div><br />
<div class="projtile" style=""><br />
<h2><a href="https://2013.igem.org/Team:Paris_Bettencourt/Project/Target">Structure Based Modelling</a></h2><br />
<p><b>SirA: </b>Using Swiss pdb we demonstrated the superimposed 3D structures of <i>Mycobacterium tuberculosis</i> SirA and <i>Escherichia coli</i> CysI highlighting their similarities and differences. Both proteins are important in their respective sulphite reduction pathways. We then predicted the effect of a small drug target based on SirA's structure. </p><br />
<br />
<p><b>FprA: </b>Using LigandScout 3.1, we searched over 8100 drug compounds from the BindingDB and Chembl databases for drugs targetting mycobacterial Ferredoxin NADP reductase (FprA). We have modelled Pyridoxine's interaction to its active site. </p><br />
</div><br />
<div class="projtile" style="margin-right:0;"><br />
<h2><a href="https://2013.igem.org/Team:Paris_Bettencourt/Project/Target">Flux Balance Analysis</a></h2><br />
<p>We used an E. coli model iJR904 obtained from BiGG database as a starting model and obtained a growth rate represented by the f value of 0.9129. We then deleted the reaction ‘SULR’ which encodes for the sulphite reduction pathway involving cysI and obtained a f value of -8.63596783409936e-13 indicating that the sulphite reduction pathway is required for growth.</p><br />
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<div id="Human_Practice"></id><br />
<h2><a href="https://2013.igem.org/wiki/index.php?title=Team:Paris_Bettencourt/Human_Practice/Overview">Human Practice</a></h2><br />
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<div class="subbox3"><br />
<div class="bkgr"><br />
<h2><a href="https://2013.igem.org/Team:Paris_Bettencourt/Human_Practice/Technology_Transfer">Technology Transfer <img height="100%" src="https://static.igem.org/mediawiki/2013/6/6d/PB_logotransferttechno.png"/></a></h2><br />
<p> An essay that addresses the issue of designing a technology aimed at "developing" countries, rather than at “developed” ones: a typical case of technology transfer.</p><br />
</div><br />
<div class="aims"><br />
<h2><a href="https://2013.igem.org/Team:Paris_Bettencourt/Human_Practice/TB_France">TB in France <img height="100%" src="https://static.igem.org/mediawiki/2013/c/cf/PB_logofrance.png"/></a></h2><br />
<p>Analysis of the social, medical and political aspects of the management of tuberculosis in France.<br />
Synthetic biology may help in many ways such as treatments, drug development, diagnostic. We also give advice on how to introduce it in clinics. </p><br />
</div><br />
</div><br />
<div class="subbox4"><br />
<div class="aims" style="height:100%"><br />
<h2><a href="https://2013.igem.org/Team:Paris_Bettencourt/Human_Practice/Gender_Study">Gender Study <img height="100%" src="https://static.igem.org/mediawiki/2013/d/da/PB_logogender.png"/></a></h2><br />
<p >A comprehensive and quantitative study of gender (in)equality in iGEM and synthetic biology. A database was gathered and statistically analysed in order to depict sex ratio of iGEM teams' students and supervisors.</p> <br />
<br><br />
<center><img width="45%" src="https://static.igem.org/mediawiki/2013/d/de/GS_Prize.png"></center><br><br />
<p style="margin-top:-10px;font-size:13px"><b>Gender balance and success in iGEM</b><br>Winning teams have a significantly higher number of women and are more Gender balanced.</p><br />
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</div> <br />
</div><br />
<br />
<div class="subbox3" style="margin-right:0"><br />
<div class="bkgr"><br />
<h2><a href="https://2013.igem.org/Team:Paris_Bettencourt/Human_Practice/TB_Facts">TB Facts <img height="100%" src="https://static.igem.org/mediawiki/2013/6/69/PB_logoTBfacts.png"/></a></h2><br />
<p> Infographics page containing TB data and facts that captures all you need to know at a glance.</p><br />
</div><br />
<div class="aims"><br />
<h2><a href="https://2013.igem.org/Team:Paris_Bettencourt/Human_Practice/TB_Gallery"> TB Gallery <img height="100%" src="https://static.igem.org/mediawiki/2013/d/de/PB_logoTBgallery.png"/><a/></h2><br />
<p> A gallery of famous historic figures who had tuberculosis, made to raise awareness to its prevalence of in the past and present .</p><br />
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<div id="Collaboration"></id><br />
<h2><a href="https://2013.igem.org/Team:Paris_Bettencourt/Collaboration">Collaboration</a></h2><br />
<div class="overbox" style="height:300px"><br />
<div class="projtile" style="height:300px"><br />
<h2><a href="https://2013.igem.org/Team:Paris_Bettencourt/SensiGEM">SensiGEM</a></h2><br />
<p>SensiGEM is the iGEM Biosensor database generated by the teams Paris Bettencourt 2013 and Calgary 2013. In this database you can find fast and easy what biosensor projects were already done by past iGEM Teams. To be able to select the projects that fit into the database, we also collaborated to compose a joint definition a biosensor.<br />
</p><br />
</div><br />
<div class="projtile" style="height:300px"><br />
<h2><a href="https://2013.igem.org/Team:Paris_Bettencourt/Collaboration">BGU iGEM Team from Israel</a></h2><br />
<p> A mutual part characterization. We characterize the promoter units produced by the lac/ara-1 promoter of cI, a repressor of their constructed kill switch. In return, BGU characterizes our TDMH biobrick protein expression levels by Western Blot. <br />
</p><br />
</div><br />
<div class="projtile" style="height:300px;margin-right:0;"><br />
<h2><a href="https://2013.igem.org/Team:Paris_Bettencourt/Collaboration">Braunschweig iGEM Team</h2><br />
<p>Idea, bibliography, and beer sharing!</p> <br />
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{{:Team:Paris_Bettencourt/footer}}</div>Idonnyahttp://2013.igem.org/Team:Paris_Bettencourt/Project/TargetTeam:Paris Bettencourt/Project/Target2013-10-29T02:04:27Z<p>Idonnya: </p>
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<div>{{:Team:Paris_Bettencourt/Wiki}}<br />
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<div id="page"><br />
<div style="width:1100px;margin:0 auto;"><br />
<img src="https://static.igem.org/mediawiki/2013/3/3a/PB_logoParis.gif" width="122px" style="position:absolute;top:40px;right:30px;"/><br />
</div><br />
<img src="https://static.igem.org/mediawiki/2013/c/c7/PB_targettitle.png" style="margin-bottom:15px"/><br />
<div class="overbox"><br />
<div class="bkgr"><br />
<h2>Background</h2><br />
<p>SirA is an essential gene in latent tuberculosis infections</p><br />
</div><br />
<div class="results"><br />
<h2>Results</h2><br />
<ul><br />
<li>Produced an <i>E. coli</i> strain which relies upon mycobacterial sirA, fprA and fdxA genes to survive in M9 minimal media</li><br />
<li>Demonstrated that <i>E. coli</i> can survive with mycobacterial sulfite reduction pathway with Flux Balance Analysis</li><br />
<li>Located drug target sites on sirA as well as identified high structural similarity between cysI and sirA through structural anaylsis</li><br />
</ul><br />
<p></p><br />
</div><br />
<div class="biocriks"><br />
<h2>BioBricks</h2><br />
<ol><br />
<li><a href="http://parts.igem.org/Part:BBa_K1137000">BBa_K1137000 (SirA)</a></li><br />
<li><a href="http://parts.igem.org/Part:BBa_K1137001">BBa_K1137001 (FprA)</a></li> <br />
<li><a href="http://parts.igem.org/Part:BBa_K1137002">BBa_K1137002 (FdxA)</a></li><br />
</ul><br />
</div><br />
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<div class="aims"><br />
<h2>Aims</h2><br />
<p>To perform a drug screen targeted at the sirA gene from mycobacteria</p><br />
</div><br />
<a href="#Introduction"><br />
<div class="hlink"><br />
<h2>Skip to Introduction</h2><br />
</div><br />
</a><br />
<a href="#Model"><br />
<div class="hlink"><br />
<h2>Skip to Modeling</h2><br />
</div><br />
</a><br />
<a href="#Design"><br />
<div class="hlink"><br />
<h2>Skip to Design</h2><br />
</div><br />
</a><br />
<a href="#Results"> <br />
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<div id="Introduction"></div><br />
<h2>Introduction</h2><br />
<div class="leftparagraph"><br />
<p> &nbsp;&nbsp;<br />
SirA is essential for <i>M. tuberculosis</i> persistence phenotype as sulfur containing amino acids are particularly sensitive to oxidative stress within the macrophage and must regularly be replaced <a href="#Reference">(Pinto <i>et al</i> 2007)</a>. Currently, there are no drug candidates that are known to specifically inhibit SirA and conventional drug screens involve do not provide information regarding the mechanism of drug action nor do compounds that inhibit exponential growth necessarily have an effect on persistent TB. We designed a working drug screen assay to specifically target the mycobacterial sulfite reductase protein SirA. To this end we cloned Ito <i>E. coli </i><span style="font-style: normal;">the sulfite reduction pathway</span> of <i>M. smegmatis</i>, a non-pathogenic mycobacterial relative of <i>M. Tuberculosis</i>. Our model overcomes the problem of long doubling time of <i>M. tuberculosis</i>. Specific inhibition of the sulfite reduction pathway is scored by comparing a drug screen of our <i>E. coli</i> construct <i>vs.</i> wild-type. Any drug candidates that have activity against both the wild-type <i>E. coli</i> and our construct are non-specific inhibitors of <i>E. coli</i> growth. However, any drug candidates that inhibit only the growth of our <i>E. coli </i>construct will be <span style="font-style: normal;">SirA</span><i> </i><span style="font-style: normal;">pathway specific.</span> <br />
</p><br />
</div><br />
<div class="rightparagraph"><br />
<a href="https://static.igem.org/mediawiki/2013/4/4f/PS_Drug_Scheme.png" target="_blank"><img src="https://static.igem.org/mediawiki/2013/4/4f/PS_Drug_Scheme.png" width="535px"/></a><br />
<p><b>Figure 1: Overview of Targeted Drug Screen Design</b></p><br />
</div><br />
<div id="Model"></div><br />
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<h2>Flux Balance Analysis of Sulfite Reduction Pathway</h2><br />
<div class="leftparagraph"><br />
<p>&nbsp;&nbsp;<br />
We used an <i>E. coli</i> model (iJR904) obtained from the <a href="http://bigg.ucsd.edu/bigg/main.pl">BiGG database</a> as a starting model to obtain wild-type growth rate (f = 0.9129 divisions/hour). We then deleted the reaction ‘SULR’ which encodes for the sulphite reduction pathway involving cysI and obtained a f= -8e-13=0 divisions/hour indicating that the sulphite reduction pathway is essential for growth. Finally we introduced two new reactions for sirA and fprA and a new species fdxA. We found that growth with the mycobacteria pathway reverts the growth phenotype back to wild-type levels (f = 0.9105 divisions/hour). We then wanted to expand our model to find new pathways that we could utilize for a targeted drug screen approach. We wrote a matlab script that finds all the essential reactions in <i>M. tuberculosis</i> and all the essential reactions in <i>E. coli</i>, and then tries to complement the essential reactions in the <i>E. coli</i> model with the essential reactions from <i>M. tuberculosis</i>. The model identified <a href="https://2013.igem.org/Team:Paris_Bettencourt/Project/Target/FBA">100 metabolic reactions</a> that we could target. Additionally, due to the modular nature of the model, it can be used to find target-able metabolic reactions in any SBML file. The Matlab scripts can be found <a href="https://2013.igem.org/File:TargetFBA.zip">here</a> and requires <a href="http://opencobra.sourceforge.net/openCOBRA/Welcome.html">Cobra Toolbox 2.0</a> to function. Please visit the FBA page for a detailed list of <a href="https://2013.igem.org/Team:Paris_Bettencourt/Project/Target/FBA">results</a>.<br />
</p><br />
</div><br />
<div class="rightparagraph"><br />
<center><a href="https://static.igem.org/mediawiki/2013/7/76/PS_FBA.png" target="_blank"><img src="https://static.igem.org/mediawiki/2013/7/76/PS_FBA.png" width="267.5px"/></a></center><br />
<p><b>Figure 2: Biomass Flux through <i>E. coli</i> and mycoSIR E. coli</b><div style="font-size: 90%">Flux balance analysis was run using Cobra Toolbox 2.0 on <i>E. coli</i> sbml model iJR904 with and without SULR reaction. Additionally an <i>E. coli</i> sbml model was built with the SULR reaction replaced with a reaction representing the mycobacterial SirA reaction and FprA reaction, as well as ferredoxin FdxA as an additional species. The Biomass flux is restored to 99.75% of the wild-type level with the synthetic mycobacterial system.</div></p><br />
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<br />
<h2>Structural Analysis of SirA</h2><br />
<div class="leftparagraph"><br />
<p>&nbsp;&nbsp;<br />
Superimposing the structures of </span><i>M.tuberculosis</i><span style="font-style: normal;"> SirA and </span><i>E.coli </i><span style="font-style: normal;">CysI reveals high homology, in particular of the active sites. Both proteins have the same symmetry (psuedo 2 fold) indicative of a common evolutionary origin. Our analysis highlighted important conserved residues, involved in substrate binding to be Arg97, Arg130, Arg166, Lys207. These positively charged residues are conserved in the sulphite/nitrite reductase family. In addition, 4 Cys residues are conserved for iron-sulphur binding. </span><br />
<p>The most profound structural differences between the two enzymes are found in the ferredoxin binding site and SirA's most C terminal residues and several surface loop regions due to deletions or insertions. A stark difference is a covalent bond formed between Cys161 (thiolate) and Tyr69 (C carbon atom) found adjacent to the redox center (Cu ions) in SirA. The covalently bound residues act as a secondary cofactor in tyrosyl radical stabilization. </p> <br />
</p><br />
</div><br />
<div class="rightparagraph"><br />
<center><a href="https://static.igem.org/mediawiki/2013/5/52/Purplecys_SirA-1.png" target="_blank"><img src="https://static.igem.org/mediawiki/2013/5/52/Purplecys_SirA-1.png" width="267.5px"/></a></center><br />
<p><b>Figure 3: The superimposed 3D protein structures of SirA and CysI.</b><div style="font-size: 90%"> 303 amino acids are involved in superimposition with an rsmd of 1.41Å. All domains and loops of CysI are coloured purple, whilst SirA is coloured according to structural similarity with CysI: Red indicates poor alignment whilst blue indicates good alignment.</div></p><br />
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<h2>Identification of potential drug target binding sites</h2><br />
<div class="leftparagraph"><br />
<p>&nbsp;&nbsp;<br />
Our structural analysis provided the basis for our drug target prediction. Using Chembl and swiss pdb, we have shown a predicted drug target site. Our calculation gives strong favour for a drug to be effective at this site. The calculation reflects the suitability of small molecules to the binding site under the Lipinski's Rule of 5.</p><br />
<p>The drug target is located at the interface of the three domains. This binding pocket exhibits a dense hydrophobic region. Our analysis targets 48 amino acids of SirA within 6Å of a modelled small drug molecule. Of these residues, only 6 amino acids are charged: His409, Asp453, Asp474, His500, Asp504 and Arg541.<br />
</p><br />
</div><br />
<div class="rightparagraph"><br />
<center><a href="https://static.igem.org/mediawiki/2013/5/59/Drug_target_withoutAA.png" target="_blank"><img src="https://static.igem.org/mediawiki/2013/5/59/Drug_target_withoutAA.png" width="267.5px"/></a></center><br />
<p><b>Figure 4 Drug target locations in SirA </b><div style="font-size: 90%">A domain located in SirA, identified as a drug target through Chembl analysis.</div></p><br />
</div><br />
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<h2>Structure based pharmacophore modelling of mycobacterial Fpra</h2><br />
<div class="leftparagraph"><br />
<p>&nbsp;&nbsp;<br />
Using LigandScount 3.1, we searched over 8100 drug compounds from the BindingDB and Chembl databases for drugs targetting mycobacterial Fpra. Our search revealed Riboflavin (Vitamin B2) and Pyridoxine to be drug targets for Fpra. We used NADP interacting with the active site as the model of the pharmacore. Results showed pyridoxin to be a competitive inhibitor to NADP. Pyridoxin is a synthetic compound currently available as a prescribed drug. </span><br />
<p>Chembl analysis of Pyridoxine (vitamin B6) show that it's properties fulfill Lipinski's criteria of being an orally active drug in humans. These properties state that any small drug molecule must have: no more than 5 H bond donors, no more 10 H bond acceptors (N or O atoms), mol mass of less than 500 dalts and octanol-water partition coefficient log P of no greater than 5).</p> <br />
</p><br />
<p>We have shown the proposed properties of Pyridoxine's interaction with Fpra as a competitive inhibitor to NADP at Fpra's active site. The key amino acids at the active site are Ala205, GLN204 and Thr208. GLN204 and Ala205 act as hydrogen bond acceptors whilst Thr208 interacts with a H via van der waals forces. Pyridoxin is a smaller, more lipid soluble molecule than NADP, thus more fitting to Lipinski's criteria. </p> <br />
</p><br />
</div><br />
<div class="rightparagraph"><br />
<center><a href="https://static.igem.org/mediawiki/2013/e/e3/Fnr_ribbons_%281%29.png" target="_blank"><img src="https://static.igem.org/mediawiki/2013/5/52/Fnr_ribbons_%281%29.png" width="267.5px"/></a></center><br />
<p><b>Figure 5: Our 3D model shows the structure of FNR where negative residues are coloured in blue, positive residues in red and NAD in purple (ball and stick representation). The key amino acids at the active site are Glu211, Gly 366, Arg 110, Arg 199, Arg 200 and Asn155. Glu211 acts as a hydrogen acceptor whilst the latter four residues act as hydrogen donors.</div></p><br />
</div><br />
<div id="Model"></div><br />
<div class="rightparagraph"><br />
<div style="clear: both;"></div><br />
<p><b>Figure 6: Comparison of NADP interaction with Fpra's active site and Pyridoxine's interaction to it's active site. </div></p><br />
</div><br />
<div id="Model"></div><br />
<div style="clear: both;"></div><br />
<div id="Design"></div><br />
<h2>Synthetic Mycobacteria Pathway</h2><br />
<div class="leftparagraph"><br />
<p> &nbsp;&nbsp;<br />
We designed a synthetic </span><i>M.smegmatis-</i><span style="font-style: normal;">derived sulfite reduction pathway containing sirA - the sulfite reductase, and two supporting genes that are required for its function in </span><i>E.coli</i><span style="font-style: normal;">: fdxA and fprA. FdxA is a mycobacterial Ferredoxin cofactor which is oxidised by SirA during the sulfite reduction reaction and FprA is a Ferredoxin-NADPH reductase use replenish the reduced Fdx pool. The genes' sequences were taken from previous work describing their expression <a href="#Reference">(Pinto <i>et al</i> 2007)</a> in </span><i>E.coli</i><span style="font-style: normal;"> for purification and in vitro characterization; we removed restriction sites and codon optimized for expression in </span><i>E. coli</i><span style="font-style: normal;">. The genes were then cloned into two Duet expression vectors, one containing sirA and one containing the supporting genesand were transformed into our knock-out mutant strains of </span><i>E. coli</i>. Data on Growth curves can be found <a href="https://2013.igem.org/Team:Paris_Bettencourt/Notebook/Drug_Screening/Monday_30th_September.html">here</a>. <br />
</p><br />
</div><br />
<div class="rightparagraph"><br />
<a href="https://static.igem.org/mediawiki/2013/8/84/TB_drug_FinalSmeg.png" target="_blank"><img src="https://static.igem.org/mediawiki/2013/8/84/TB_drug_FinalSmeg.png" width="535px"/></a><br />
<p><b>Figure 5: Growth curves of <i>E. coli</i> mycoSIR</b> <div style="font-size: 90%">BL21 (DE3) ΔcysI containing the MycoSIR pathway (MycoSIR <i>E. coli</i>) were grown in liquid minimal media containing Various concentration of IPTG. (A) Replicates of each strain were measured for absorbance in a spectrophotometer every 10 minutes for 14 hours. Growth was observed for the WT BL21 <i>E. coli</i>, (blue), and the MycoSIR <i>E. coli</i> (red). No growth was detected for uninduced MycoSIR <i>E. coli</i> (purple) or for the BL21 (DE3) ΔcysI that did not contain the synthetic pathway (Orange) . (B) Mean Final ODs of all replicates, measured after 14 hours of growth. Growth was detected in zmSIR <i>E. coli</i> and WT BL21 but not in uninduced zmSIR strain.</div></p> <br />
</div><br />
<div style="clear: both;"></div><br />
<h2>Creation of Knock out Mutants</h2><br />
<div class="leftparagraph"><br />
<p>&nbsp;&nbsp;<br />
We prepared two strains of <i>E. coli</i> which have the sulfite reduction pathway deleted: BL21 (DE3) <i>ΔCysI Δfpr ΔydbK</i> and BL21 (AI) <i>ΔCysI</i>. CysI is responsible for sulfite reduction in <i>E. coli</i>, while <i>fpr and ydbK</i> are two non-essential genes that consume ferredoxin. These two genes are deleted, as sulfite reduction in mycobacteria is ferredoxin dependent in comparison to<i> E. coli</i> in which it is NADPH dependant. These genes were also removed to ensure that they do not interfere with our system. <br />
</div><br />
<div class="rightparagraph"><br />
</div><br />
<div style="clear: both;"></div><br />
<h2>Synthetic Corn Pathway</h2><br />
<div class="leftparagraph"><br />
<p>&nbsp;&nbsp;<br />
Additionally we prototyped the system with a reconstruction of a sulphite reduction pathway previously designed and published by the silver group <a href="#Reference">(2011 Barstow et al)</a>. In place of CysI, a corn (Zea mays) derived sulfite reductase (zmSIR) was used. Two additional genes were included: Spinach ferredoxin (soFD), and corn derived ferredoxin NADP+ reductase (zmFNR). These genes, respectively, are required for production of the ferredoxin cofactor and the NADP+ ferredoxin reductase and are required for sulfite reductase (zmSIR) to function within <i>E. coli</i>.<br />
</p><br />
</div><br />
<div class="rightparagraph"><br />
<a href="https://static.igem.org/mediawiki/2013/a/a2/PB_final_Corn.png" target="_blank"><img src="https://static.igem.org/mediawiki/2013/a/a2/PB_final_Corn.png" width="535px"/></a><br />
<p><b>Figure 6: Growth curves of <i>E. coli</i> maizeSIR</b><div style="font-size: 90%"> BL21 (DE3) ΔcysI containing the MaizeSIR pathway (MaizeSIR <i>E. coli</i>) were grown in liquid minimal media containing Various concentration of IPTG. (A) Replicates of each strain were measured for absorbance in a spectrophotometer every 10 minutes for 14 hours. Growth was observed for the WT BL21 <i>E. coli</i>, (blue), and the MaizeSIR <i>E. coli</i> (red). No growth was detected for uninduced MaizeSIR <i>E. coli</i> (purple) or for the BL21 (DE3) ΔcysI that did not contain the synthetic pathway (Orange) . (B) Mean Final ODs of all replicates, measured after 14 hours of growth. Growth was detected in zmSIR <i>E. coli</i> and WT BL21 but not in uninduced zmSIR strain.</div></p> <br />
</div><br />
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<div id="Results"></div><br />
<h2>Results</h2><br />
<div class="leftparagraph"><br />
<p>&nbsp;&nbsp;<br />
Upon successful cloning of the three genes into our <i>E. coli</i> deletion strain, we continued to confirm that all three genes are required for growth on minimal media. Our two synthetic pathways were found to rescue growth on a sulfurless amino acid supplemented minimal media. We hope that this technique of using synthetic biology to overcome problems faced in naturally occurring systems will be both a large boon to the pursuit of finding novel drug candidates in <i>M. tuberculosis</i> and more broadly as this technique can be used for high-throughput screening of any pathway that can be constructed to be essential for growth in <i>E. coli</i>.<br />
</p><br />
</div><br />
<div class="rightparagraph"><br />
<a href="https://static.igem.org/mediawiki/2013/8/85/PS_D_Figure1_plate.png" target="_blank"><img src="https://static.igem.org/mediawiki/2013/8/85/PS_D_Figure1_plate.png" width="535px"/></a><br />
<p><b>Figure 7: Growth of zmSIR <i>E. coli</i> on minimal media.</b> <div style="font-size: 90%">BL21 (DE3) ΔcysI cells transformed with 1, 2 and 3 genes of the 3-gene zmSIR synthetic pathway were grown for 24 hours on minimal media supplemented with 25 uM IPTG (see methods), along with a WT BL21 (DE3) serving as a negative control, and an untransformed BL21 (DE3) ΔcysI, as negative control. Rescue of growth required all genes of the synthetic pathway (SIR, FNR and FD). </div></p><br />
</div><br />
<div style="clear: both;"></div><br />
<br />
<h2>Z-score</h2><br />
<div class="leftparagraph"><br />
<p>&nbsp;&nbsp;<br />
The Z-score is a statistical measurement aimed at assessing the "hit effect" in a drug screen high throughput screening. It is a commonly used measurement that shows how well did the drug effect the growth of the assay strain and how significant is the decrease in growth.</p><br />
<br />
<p>&nbsp;&nbsp;<br />
To calculate the Z-score we used our experimental <i>E. coli</i> strain BL21 (AI) ΔcysI that carries all three genes of the synthetic pathway (sirA, fprA, fdxA). We grew it in the M9 minimal media supplemented with amino acid sulfur dropout powder, in a 96 well plate. Four of the wells were "spiked" with antibiotics (Amp, Gent, Kan, and Spect). </p><br />
</div><br />
<div class="rightparagraph"><br />
<p>&nbsp;&nbsp;<br />
This is basically a simulation of the drug screen but without the actual drugs. Only the drug screen controls are used: growth in M9 as a negative control (no drugs) and growth in M9 + antibiotics as a positive control (a sure hit). Then we compared the distribution of the growth in the negative control with the distribution of growth in the positive control. The Z-score shows how many standard deviations away from each other are the means of the two distributions.</p><br />
<p><b>Our Z-score is: -10.2.</b></div></p><br />
<br />
<div style="clear: both;"></div><br />
<br />
<h2>Z-factor</h2><br />
<div class="leftparagraph"><br />
<p>&nbsp;&nbsp;<br />
Z-factor is a measurement complementary to the Z-score. It measures the assay's quality based on the same data extracted from the same experiment made for the Z-score. This calculation gives an estimation of how far the negative controls are from the positive controls. It is a comparison of the two distributions which assumes that both distributions are normal and calculate how far 99% of the data points of each distribution are from each other.</p><br />
<br />
</div><br />
<div class="rightparagraph"><br />
<p>&nbsp;&nbsp;<br />
Z-factor is given on a scale from 0 to 1. Scores between 0.5 and 1 show that the assay is good and will enable testing in High throughput screens.</p><br />
<p><b>Our Z-factor score is 0.58.</b></div></p><br />
</div><br />
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<div id="Reference"></div><br />
<h2>Literature</h2><br />
<div class="leftparagraph"><br />
<ul><br />
<li>Global Alliance for TB Drug Development, Tuberculosis. Scientific blueprint for tuberculosis drug development, Tuberculosis (Edinb) 81 Suppl 1, 1–52 (2001).</li><br />
<br />
<li>World Health Organization, Global Tuberculosis Report 2012 (2012).</li><br />
<br />
<li>K. Raman, K. Yeturu, N. Chandra, targetTB: A target identification pipeline for Mycobacterium tuberculosis through an interactome, reactome and genome-scale structural analysis, BMC Syst Biol 2, 109 (2008).</li><br />
<br />
<li>R. Pinto, J. S. Harrison, T. Hsu, W. R. Jacobs, T. S. Leyh, Sulfite Reduction in Mycobacteria, Journal of Bacteriology 189, 6714–6722 (2007).</li><br />
<br />
<li>B. Barstow C. M. Agapakis, P. M. Boyle, G. Grandl, P. A. Silver, E. H. Wintermute, A synthetic system links FeFe-hydrogenases to essential E. coli sulfur metabolism, J Biol Eng 5, 7 (2011).</li><br />
</ul><br />
<br />
</div><br />
</div><br />
<div class="rightparagraph"><br />
<ul><br />
<li>Schellenberger J, Que R, Fleming RMT, Thiele I, Orth JD, Feist AM, Zielinski DC, Bordbar A, Lewis NE, Rahmanian S, Kang J, Hyduke DR, Palsson BØ. 2011 Quantitative prediction of cellular metabolism with constraint-based models: the COBRA Toolbox v2.0. Nature Protocols 6:1290-1307.</li><br />
<br />
<li>Schellenberger, J., Park, J. O., Conrad, T. C., and Palsson, B. Ø., BiGG: a Biochemical Genetic and Genomic knowledgebase of large scale metabolic reconstructions, BMC Bioinformatics, 11:213, (2010).</li><br />
<br />
<li>S. G. Franzblau et al., Comprehensive analysis of methods used for the evaluation of compounds against Mycobacterium tuberculosis, Tuberculosis 92, 453–488 (2012).</li><br />
<br />
<li>D. J. Payne, M. N. Gwynn, D. J. Holmes, D. L. Pompliano, Drugs for bad bugs: confronting the challenges of antibacterial discovery, Nat Rev Drug Discov 6, 29–40 (2006).</li><br />
<br />
<li>M. Nakayama, T. Akashi, T. Hase, Plant sulfite reductase: molecular structure, catalytic function and interaction with ferredoxin, J. Inorg. Biochem. 82, 27–32 (2000).</li><br />
</ul><br />
<br />
</div><br />
<div style="clear: both;"></div><br />
<h2>Attributions</h2><br />
<p>Strains NEBTurbo, BL21 (DE3) KO20, BL21 AI were provided by INSERM U1001.</p><br />
<p>Plasmids pET Duet, pACYC Duet, pACYC zmSIR, pACYC soFD zmSIR, pCDF FNR were provided by INSERM U1001.</p><br />
<p>Genes msSirA, msFprA, msFdxA were synthesized by IDT.</p><br />
<p>Project was designed by Idonnya Aghoghogbe, Yonatan Zegman, Matthew Deyell and Edwin Wintermute. All experiments and modelling were performed by Idonnya Aghoghogbe, Yonatan Zegman, Matthew Deyell. </p><br />
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{{:Team:Paris_Bettencourt/footer}}</div>Idonnyahttp://2013.igem.org/File:Fnr_ribbons_(1).pngFile:Fnr ribbons (1).png2013-10-29T02:02:45Z<p>Idonnya: Our 3D model shows the structure of FNR where negative residues are coloured in blue, positive residues in red and NAD in purple (ball and stick representation). The key amino acids at the active site are Glu211, Gly 366, Arg 110, Arg 199, Arg 200 and Asn</p>
<hr />
<div>Our 3D model shows the structure of FNR where negative residues are coloured in blue, positive residues in red and NAD in purple (ball and stick representation). The key amino acids at the active site are Glu211, Gly 366, Arg 110, Arg 199, Arg 200 and Asn155. Glu211 acts as a hydrogen acceptor whilst the latter four residues act as hydrogen donors.</div>Idonnyahttp://2013.igem.org/Team:Paris_Bettencourt/Project/TargetTeam:Paris Bettencourt/Project/Target2013-10-29T01:58:09Z<p>Idonnya: </p>
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<div>{{:Team:Paris_Bettencourt/Wiki}}<br />
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<div id="page"><br />
<div style="width:1100px;margin:0 auto;"><br />
<img src="https://static.igem.org/mediawiki/2013/3/3a/PB_logoParis.gif" width="122px" style="position:absolute;top:40px;right:30px;"/><br />
</div><br />
<img src="https://static.igem.org/mediawiki/2013/c/c7/PB_targettitle.png" style="margin-bottom:15px"/><br />
<div class="overbox"><br />
<div class="bkgr"><br />
<h2>Background</h2><br />
<p>SirA is an essential gene in latent tuberculosis infections</p><br />
</div><br />
<div class="results"><br />
<h2>Results</h2><br />
<ul><br />
<li>Produced an <i>E. coli</i> strain which relies upon mycobacterial sirA, fprA and fdxA genes to survive in M9 minimal media</li><br />
<li>Demonstrated that <i>E. coli</i> can survive with mycobacterial sulfite reduction pathway with Flux Balance Analysis</li><br />
<li>Located drug target sites on sirA as well as identified high structural similarity between cysI and sirA through structural anaylsis</li><br />
</ul><br />
<p></p><br />
</div><br />
<div class="biocriks"><br />
<h2>BioBricks</h2><br />
<ol><br />
<li><a href="http://parts.igem.org/Part:BBa_K1137000">BBa_K1137000 (SirA)</a></li><br />
<li><a href="http://parts.igem.org/Part:BBa_K1137001">BBa_K1137001 (FprA)</a></li> <br />
<li><a href="http://parts.igem.org/Part:BBa_K1137002">BBa_K1137002 (FdxA)</a></li><br />
</ul><br />
</div><br />
<div style="clear: both;"></div><br />
<div class="aims"><br />
<h2>Aims</h2><br />
<p>To perform a drug screen targeted at the sirA gene from mycobacteria</p><br />
</div><br />
<a href="#Introduction"><br />
<div class="hlink"><br />
<h2>Skip to Introduction</h2><br />
</div><br />
</a><br />
<a href="#Model"><br />
<div class="hlink"><br />
<h2>Skip to Modeling</h2><br />
</div><br />
</a><br />
<a href="#Design"><br />
<div class="hlink"><br />
<h2>Skip to Design</h2><br />
</div><br />
</a><br />
<a href="#Results"> <br />
<div class="hlink" style="margin-right:0px"><br />
<h2>Skip to Results</h2><br />
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</div><br />
<div id="Introduction"></div><br />
<h2>Introduction</h2><br />
<div class="leftparagraph"><br />
<p> &nbsp;&nbsp;<br />
SirA is essential for <i>M. tuberculosis</i> persistence phenotype as sulfur containing amino acids are particularly sensitive to oxidative stress within the macrophage and must regularly be replaced <a href="#Reference">(Pinto <i>et al</i> 2007)</a>. Currently, there are no drug candidates that are known to specifically inhibit SirA and conventional drug screens involve do not provide information regarding the mechanism of drug action nor do compounds that inhibit exponential growth necessarily have an effect on persistent TB. We designed a working drug screen assay to specifically target the mycobacterial sulfite reductase protein SirA. To this end we cloned Ito <i>E. coli </i><span style="font-style: normal;">the sulfite reduction pathway</span> of <i>M. smegmatis</i>, a non-pathogenic mycobacterial relative of <i>M. Tuberculosis</i>. Our model overcomes the problem of long doubling time of <i>M. tuberculosis</i>. Specific inhibition of the sulfite reduction pathway is scored by comparing a drug screen of our <i>E. coli</i> construct <i>vs.</i> wild-type. Any drug candidates that have activity against both the wild-type <i>E. coli</i> and our construct are non-specific inhibitors of <i>E. coli</i> growth. However, any drug candidates that inhibit only the growth of our <i>E. coli </i>construct will be <span style="font-style: normal;">SirA</span><i> </i><span style="font-style: normal;">pathway specific.</span> <br />
</p><br />
</div><br />
<div class="rightparagraph"><br />
<a href="https://static.igem.org/mediawiki/2013/4/4f/PS_Drug_Scheme.png" target="_blank"><img src="https://static.igem.org/mediawiki/2013/4/4f/PS_Drug_Scheme.png" width="535px"/></a><br />
<p><b>Figure 1: Overview of Targeted Drug Screen Design</b></p><br />
</div><br />
<div id="Model"></div><br />
<div style="clear: both;"></div><br />
<h2>Flux Balance Analysis of Sulfite Reduction Pathway</h2><br />
<div class="leftparagraph"><br />
<p>&nbsp;&nbsp;<br />
We used an <i>E. coli</i> model (iJR904) obtained from the <a href="http://bigg.ucsd.edu/bigg/main.pl">BiGG database</a> as a starting model to obtain wild-type growth rate (f = 0.9129 divisions/hour). We then deleted the reaction ‘SULR’ which encodes for the sulphite reduction pathway involving cysI and obtained a f= -8e-13=0 divisions/hour indicating that the sulphite reduction pathway is essential for growth. Finally we introduced two new reactions for sirA and fprA and a new species fdxA. We found that growth with the mycobacteria pathway reverts the growth phenotype back to wild-type levels (f = 0.9105 divisions/hour). We then wanted to expand our model to find new pathways that we could utilize for a targeted drug screen approach. We wrote a matlab script that finds all the essential reactions in <i>M. tuberculosis</i> and all the essential reactions in <i>E. coli</i>, and then tries to complement the essential reactions in the <i>E. coli</i> model with the essential reactions from <i>M. tuberculosis</i>. The model identified <a href="https://2013.igem.org/Team:Paris_Bettencourt/Project/Target/FBA">100 metabolic reactions</a> that we could target. Additionally, due to the modular nature of the model, it can be used to find target-able metabolic reactions in any SBML file. The Matlab scripts can be found <a href="https://2013.igem.org/File:TargetFBA.zip">here</a> and requires <a href="http://opencobra.sourceforge.net/openCOBRA/Welcome.html">Cobra Toolbox 2.0</a> to function. Please visit the FBA page for a detailed list of <a href="https://2013.igem.org/Team:Paris_Bettencourt/Project/Target/FBA">results</a>.<br />
</p><br />
</div><br />
<div class="rightparagraph"><br />
<center><a href="https://static.igem.org/mediawiki/2013/7/76/PS_FBA.png" target="_blank"><img src="https://static.igem.org/mediawiki/2013/7/76/PS_FBA.png" width="267.5px"/></a></center><br />
<p><b>Figure 2: Biomass Flux through <i>E. coli</i> and mycoSIR E. coli</b><div style="font-size: 90%">Flux balance analysis was run using Cobra Toolbox 2.0 on <i>E. coli</i> sbml model iJR904 with and without SULR reaction. Additionally an <i>E. coli</i> sbml model was built with the SULR reaction replaced with a reaction representing the mycobacterial SirA reaction and FprA reaction, as well as ferredoxin FdxA as an additional species. The Biomass flux is restored to 99.75% of the wild-type level with the synthetic mycobacterial system.</div></p><br />
</div><br />
<div id="Model"></div><br />
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<br />
<h2>Structural Analysis of SirA</h2><br />
<div class="leftparagraph"><br />
<p>&nbsp;&nbsp;<br />
Superimposing the structures of </span><i>M.tuberculosis</i><span style="font-style: normal;"> SirA and </span><i>E.coli </i><span style="font-style: normal;">CysI reveals high homology, in particular of the active sites. Both proteins have the same symmetry (psuedo 2 fold) indicative of a common evolutionary origin. Our analysis highlighted important conserved residues, involved in substrate binding to be Arg97, Arg130, Arg166, Lys207. These positively charged residues are conserved in the sulphite/nitrite reductase family. In addition, 4 Cys residues are conserved for iron-sulphur binding. </span><br />
<p>The most profound structural differences between the two enzymes are found in the ferredoxin binding site and SirA's most C terminal residues and several surface loop regions due to deletions or insertions. A stark difference is a covalent bond formed between Cys161 (thiolate) and Tyr69 (C carbon atom) found adjacent to the redox center (Cu ions) in SirA. The covalently bound residues act as a secondary cofactor in tyrosyl radical stabilization. </p> <br />
</p><br />
</div><br />
<div class="rightparagraph"><br />
<center><a href="https://static.igem.org/mediawiki/2013/5/52/Purplecys_SirA-1.png" target="_blank"><img src="https://static.igem.org/mediawiki/2013/5/52/Purplecys_SirA-1.png" width="267.5px"/></a></center><br />
<p><b>Figure 3: The superimposed 3D protein structures of SirA and CysI.</b><div style="font-size: 90%"> 303 amino acids are involved in superimposition with an rsmd of 1.41Å. All domains and loops of CysI are coloured purple, whilst SirA is coloured according to structural similarity with CysI: Red indicates poor alignment whilst blue indicates good alignment.</div></p><br />
</div><br />
<div id="Model"></div><br />
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<h2>Identification of potential drug target binding sites</h2><br />
<div class="leftparagraph"><br />
<p>&nbsp;&nbsp;<br />
Our structural analysis provided the basis for our drug target prediction. Using Chembl and swiss pdb, we have shown a predicted drug target site. Our calculation gives strong favour for a drug to be effective at this site. The calculation reflects the suitability of small molecules to the binding site under the Lipinski's Rule of 5.</p><br />
<p>The drug target is located at the interface of the three domains. This binding pocket exhibits a dense hydrophobic region. Our analysis targets 48 amino acids of SirA within 6Å of a modelled small drug molecule. Of these residues, only 6 amino acids are charged: His409, Asp453, Asp474, His500, Asp504 and Arg541.<br />
</p><br />
</div><br />
<div class="rightparagraph"><br />
<center><a href="https://static.igem.org/mediawiki/2013/5/59/Drug_target_withoutAA.png" target="_blank"><img src="https://static.igem.org/mediawiki/2013/5/59/Drug_target_withoutAA.png" width="267.5px"/></a></center><br />
<p><b>Figure 4 Drug target locations in SirA </b><div style="font-size: 90%">A domain located in SirA, identified as a drug target through Chembl analysis.</div></p><br />
</div><br />
<div style="clear: both;"></div><br />
<h2>Structure based pharmacophore modelling of mycobacterial Fpra</h2><br />
<div class="leftparagraph"><br />
<p>&nbsp;&nbsp;<br />
Using LigandScount 3.1, we searched over 8100 drug compounds from the BindingDB and Chembl databases for drugs targetting mycobacterial Fpra. Our search revealed Riboflavin (Vitamin B2) and Pyridoxine to be drug targets for Fpra. We used NADP interacting with the active site as the model of the pharmacore. Results showed pyridoxin to be a competitive inhibitor to NADP. Pyridoxin is a synthetic compound currently available as a prescribed drug. </span><br />
<p>Chembl analysis of Pyridoxine (vitamin B6) show that it's properties fulfill Lipinski's criteria of being an orally active drug in humans. These properties state that any small drug molecule must have: no more than 5 H bond donors, no more 10 H bond acceptors (N or O atoms), mol mass of less than 500 dalts and octanol-water partition coefficient log P of no greater than 5).</p> <br />
</p><br />
<p>We have shown the proposed properties of Pyridoxine's interaction with Fpra as a competitive inhibitor to NADP at Fpra's active site. The key amino acids at the active site are Ala205, GLN204 and Thr208. GLN204 and Ala205 act as hydrogen bond acceptors whilst Thr208 interacts with a H via van der waals forces. Pyridoxin is a smaller, more lipid soluble molecule than NADP, thus more fitting to Lipinski's criteria. </p> <br />
</p><br />
</div><br />
<div class="rightparagraph"><br />
<center><a href="https://static.igem.org/mediawiki/2013/6/60/Fnr_ribbons.png" target="_blank"><img src="https://static.igem.org/mediawiki/2013/5/52/Fnr_ribbons.png" width="267.5px"/></a></center><br />
<p><b>Figure 5: Our 3D model shows the structure of FNR where negative residues are coloured in blue, positive residues in red and NAD in purple (ball and stick representation). The key amino acids at the active site are Glu211, Gly 366, Arg 110, Arg 199, Arg 200 and Asn155. Glu211 acts as a hydrogen acceptor whilst the latter four residues act as hydrogen donors.</div></p><br />
</div><br />
<div id="Model"></div><br />
<div class="rightparagraph"><br />
<div style="clear: both;"></div><br />
<p><b>Figure 6: Comparison of NADP interaction with Fpra's active site and Pyridoxine's interaction to it's active site. </div></p><br />
</div><br />
<div id="Model"></div><br />
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<div id="Design"></div><br />
<h2>Synthetic Mycobacteria Pathway</h2><br />
<div class="leftparagraph"><br />
<p> &nbsp;&nbsp;<br />
We designed a synthetic </span><i>M.smegmatis-</i><span style="font-style: normal;">derived sulfite reduction pathway containing sirA - the sulfite reductase, and two supporting genes that are required for its function in </span><i>E.coli</i><span style="font-style: normal;">: fdxA and fprA. FdxA is a mycobacterial Ferredoxin cofactor which is oxidised by SirA during the sulfite reduction reaction and FprA is a Ferredoxin-NADPH reductase use replenish the reduced Fdx pool. The genes' sequences were taken from previous work describing their expression <a href="#Reference">(Pinto <i>et al</i> 2007)</a> in </span><i>E.coli</i><span style="font-style: normal;"> for purification and in vitro characterization; we removed restriction sites and codon optimized for expression in </span><i>E. coli</i><span style="font-style: normal;">. The genes were then cloned into two Duet expression vectors, one containing sirA and one containing the supporting genesand were transformed into our knock-out mutant strains of </span><i>E. coli</i>. Data on Growth curves can be found <a href="https://2013.igem.org/Team:Paris_Bettencourt/Notebook/Drug_Screening/Monday_30th_September.html">here</a>. <br />
</p><br />
</div><br />
<div class="rightparagraph"><br />
<a href="https://static.igem.org/mediawiki/2013/8/84/TB_drug_FinalSmeg.png" target="_blank"><img src="https://static.igem.org/mediawiki/2013/8/84/TB_drug_FinalSmeg.png" width="535px"/></a><br />
<p><b>Figure 5: Growth curves of <i>E. coli</i> mycoSIR</b> <div style="font-size: 90%">BL21 (DE3) ΔcysI containing the MycoSIR pathway (MycoSIR <i>E. coli</i>) were grown in liquid minimal media containing Various concentration of IPTG. (A) Replicates of each strain were measured for absorbance in a spectrophotometer every 10 minutes for 14 hours. Growth was observed for the WT BL21 <i>E. coli</i>, (blue), and the MycoSIR <i>E. coli</i> (red). No growth was detected for uninduced MycoSIR <i>E. coli</i> (purple) or for the BL21 (DE3) ΔcysI that did not contain the synthetic pathway (Orange) . (B) Mean Final ODs of all replicates, measured after 14 hours of growth. Growth was detected in zmSIR <i>E. coli</i> and WT BL21 but not in uninduced zmSIR strain.</div></p> <br />
</div><br />
<div style="clear: both;"></div><br />
<h2>Creation of Knock out Mutants</h2><br />
<div class="leftparagraph"><br />
<p>&nbsp;&nbsp;<br />
We prepared two strains of <i>E. coli</i> which have the sulfite reduction pathway deleted: BL21 (DE3) <i>ΔCysI Δfpr ΔydbK</i> and BL21 (AI) <i>ΔCysI</i>. CysI is responsible for sulfite reduction in <i>E. coli</i>, while <i>fpr and ydbK</i> are two non-essential genes that consume ferredoxin. These two genes are deleted, as sulfite reduction in mycobacteria is ferredoxin dependent in comparison to<i> E. coli</i> in which it is NADPH dependant. These genes were also removed to ensure that they do not interfere with our system. <br />
</div><br />
<div class="rightparagraph"><br />
</div><br />
<div style="clear: both;"></div><br />
<h2>Synthetic Corn Pathway</h2><br />
<div class="leftparagraph"><br />
<p>&nbsp;&nbsp;<br />
Additionally we prototyped the system with a reconstruction of a sulphite reduction pathway previously designed and published by the silver group <a href="#Reference">(2011 Barstow et al)</a>. In place of CysI, a corn (Zea mays) derived sulfite reductase (zmSIR) was used. Two additional genes were included: Spinach ferredoxin (soFD), and corn derived ferredoxin NADP+ reductase (zmFNR). These genes, respectively, are required for production of the ferredoxin cofactor and the NADP+ ferredoxin reductase and are required for sulfite reductase (zmSIR) to function within <i>E. coli</i>.<br />
</p><br />
</div><br />
<div class="rightparagraph"><br />
<a href="https://static.igem.org/mediawiki/2013/a/a2/PB_final_Corn.png" target="_blank"><img src="https://static.igem.org/mediawiki/2013/a/a2/PB_final_Corn.png" width="535px"/></a><br />
<p><b>Figure 6: Growth curves of <i>E. coli</i> maizeSIR</b><div style="font-size: 90%"> BL21 (DE3) ΔcysI containing the MaizeSIR pathway (MaizeSIR <i>E. coli</i>) were grown in liquid minimal media containing Various concentration of IPTG. (A) Replicates of each strain were measured for absorbance in a spectrophotometer every 10 minutes for 14 hours. Growth was observed for the WT BL21 <i>E. coli</i>, (blue), and the MaizeSIR <i>E. coli</i> (red). No growth was detected for uninduced MaizeSIR <i>E. coli</i> (purple) or for the BL21 (DE3) ΔcysI that did not contain the synthetic pathway (Orange) . (B) Mean Final ODs of all replicates, measured after 14 hours of growth. Growth was detected in zmSIR <i>E. coli</i> and WT BL21 but not in uninduced zmSIR strain.</div></p> <br />
</div><br />
<div style="clear: both;"></div><br />
<div id="Results"></div><br />
<h2>Results</h2><br />
<div class="leftparagraph"><br />
<p>&nbsp;&nbsp;<br />
Upon successful cloning of the three genes into our <i>E. coli</i> deletion strain, we continued to confirm that all three genes are required for growth on minimal media. Our two synthetic pathways were found to rescue growth on a sulfurless amino acid supplemented minimal media. We hope that this technique of using synthetic biology to overcome problems faced in naturally occurring systems will be both a large boon to the pursuit of finding novel drug candidates in <i>M. tuberculosis</i> and more broadly as this technique can be used for high-throughput screening of any pathway that can be constructed to be essential for growth in <i>E. coli</i>.<br />
</p><br />
</div><br />
<div class="rightparagraph"><br />
<a href="https://static.igem.org/mediawiki/2013/8/85/PS_D_Figure1_plate.png" target="_blank"><img src="https://static.igem.org/mediawiki/2013/8/85/PS_D_Figure1_plate.png" width="535px"/></a><br />
<p><b>Figure 7: Growth of zmSIR <i>E. coli</i> on minimal media.</b> <div style="font-size: 90%">BL21 (DE3) ΔcysI cells transformed with 1, 2 and 3 genes of the 3-gene zmSIR synthetic pathway were grown for 24 hours on minimal media supplemented with 25 uM IPTG (see methods), along with a WT BL21 (DE3) serving as a negative control, and an untransformed BL21 (DE3) ΔcysI, as negative control. Rescue of growth required all genes of the synthetic pathway (SIR, FNR and FD). </div></p><br />
</div><br />
<div style="clear: both;"></div><br />
<br />
<h2>Z-score</h2><br />
<div class="leftparagraph"><br />
<p>&nbsp;&nbsp;<br />
The Z-score is a statistical measurement aimed at assessing the "hit effect" in a drug screen high throughput screening. It is a commonly used measurement that shows how well did the drug effect the growth of the assay strain and how significant is the decrease in growth.</p><br />
<br />
<p>&nbsp;&nbsp;<br />
To calculate the Z-score we used our experimental <i>E. coli</i> strain BL21 (AI) ΔcysI that carries all three genes of the synthetic pathway (sirA, fprA, fdxA). We grew it in the M9 minimal media supplemented with amino acid sulfur dropout powder, in a 96 well plate. Four of the wells were "spiked" with antibiotics (Amp, Gent, Kan, and Spect). </p><br />
</div><br />
<div class="rightparagraph"><br />
<p>&nbsp;&nbsp;<br />
This is basically a simulation of the drug screen but without the actual drugs. Only the drug screen controls are used: growth in M9 as a negative control (no drugs) and growth in M9 + antibiotics as a positive control (a sure hit). Then we compared the distribution of the growth in the negative control with the distribution of growth in the positive control. The Z-score shows how many standard deviations away from each other are the means of the two distributions.</p><br />
<p><b>Our Z-score is: -10.2.</b></div></p><br />
<br />
<div style="clear: both;"></div><br />
<br />
<h2>Z-factor</h2><br />
<div class="leftparagraph"><br />
<p>&nbsp;&nbsp;<br />
Z-factor is a measurement complementary to the Z-score. It measures the assay's quality based on the same data extracted from the same experiment made for the Z-score. This calculation gives an estimation of how far the negative controls are from the positive controls. It is a comparison of the two distributions which assumes that both distributions are normal and calculate how far 99% of the data points of each distribution are from each other.</p><br />
<br />
</div><br />
<div class="rightparagraph"><br />
<p>&nbsp;&nbsp;<br />
Z-factor is given on a scale from 0 to 1. Scores between 0.5 and 1 show that the assay is good and will enable testing in High throughput screens.</p><br />
<p><b>Our Z-factor score is 0.58.</b></div></p><br />
</div><br />
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<div id="Reference"></div><br />
<h2>Literature</h2><br />
<div class="leftparagraph"><br />
<ul><br />
<li>Global Alliance for TB Drug Development, Tuberculosis. Scientific blueprint for tuberculosis drug development, Tuberculosis (Edinb) 81 Suppl 1, 1–52 (2001).</li><br />
<br />
<li>World Health Organization, Global Tuberculosis Report 2012 (2012).</li><br />
<br />
<li>K. Raman, K. Yeturu, N. Chandra, targetTB: A target identification pipeline for Mycobacterium tuberculosis through an interactome, reactome and genome-scale structural analysis, BMC Syst Biol 2, 109 (2008).</li><br />
<br />
<li>R. Pinto, J. S. Harrison, T. Hsu, W. R. Jacobs, T. S. Leyh, Sulfite Reduction in Mycobacteria, Journal of Bacteriology 189, 6714–6722 (2007).</li><br />
<br />
<li>B. Barstow C. M. Agapakis, P. M. Boyle, G. Grandl, P. A. Silver, E. H. Wintermute, A synthetic system links FeFe-hydrogenases to essential E. coli sulfur metabolism, J Biol Eng 5, 7 (2011).</li><br />
</ul><br />
<br />
</div><br />
</div><br />
<div class="rightparagraph"><br />
<ul><br />
<li>Schellenberger J, Que R, Fleming RMT, Thiele I, Orth JD, Feist AM, Zielinski DC, Bordbar A, Lewis NE, Rahmanian S, Kang J, Hyduke DR, Palsson BØ. 2011 Quantitative prediction of cellular metabolism with constraint-based models: the COBRA Toolbox v2.0. Nature Protocols 6:1290-1307.</li><br />
<br />
<li>Schellenberger, J., Park, J. O., Conrad, T. C., and Palsson, B. Ø., BiGG: a Biochemical Genetic and Genomic knowledgebase of large scale metabolic reconstructions, BMC Bioinformatics, 11:213, (2010).</li><br />
<br />
<li>S. G. Franzblau et al., Comprehensive analysis of methods used for the evaluation of compounds against Mycobacterium tuberculosis, Tuberculosis 92, 453–488 (2012).</li><br />
<br />
<li>D. J. Payne, M. N. Gwynn, D. J. Holmes, D. L. Pompliano, Drugs for bad bugs: confronting the challenges of antibacterial discovery, Nat Rev Drug Discov 6, 29–40 (2006).</li><br />
<br />
<li>M. Nakayama, T. Akashi, T. Hase, Plant sulfite reductase: molecular structure, catalytic function and interaction with ferredoxin, J. Inorg. Biochem. 82, 27–32 (2000).</li><br />
</ul><br />
<br />
</div><br />
<div style="clear: both;"></div><br />
<h2>Attributions</h2><br />
<p>Strains NEBTurbo, BL21 (DE3) KO20, BL21 AI were provided by INSERM U1001.</p><br />
<p>Plasmids pET Duet, pACYC Duet, pACYC zmSIR, pACYC soFD zmSIR, pCDF FNR were provided by INSERM U1001.</p><br />
<p>Genes msSirA, msFprA, msFdxA were synthesized by IDT.</p><br />
<p>Project was designed by Idonnya Aghoghogbe, Yonatan Zegman, Matthew Deyell and Edwin Wintermute. All experiments and modelling were performed by Idonnya Aghoghogbe, Yonatan Zegman, Matthew Deyell. </p><br />
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{{:Team:Paris_Bettencourt/footer}}</div>Idonnyahttp://2013.igem.org/Team:Paris_Bettencourt/Project/TargetTeam:Paris Bettencourt/Project/Target2013-10-29T01:55:31Z<p>Idonnya: </p>
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<div>{{:Team:Paris_Bettencourt/Wiki}}<br />
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<div id="page"><br />
<div style="width:1100px;margin:0 auto;"><br />
<img src="https://static.igem.org/mediawiki/2013/3/3a/PB_logoParis.gif" width="122px" style="position:absolute;top:40px;right:30px;"/><br />
</div><br />
<img src="https://static.igem.org/mediawiki/2013/c/c7/PB_targettitle.png" style="margin-bottom:15px"/><br />
<div class="overbox"><br />
<div class="bkgr"><br />
<h2>Background</h2><br />
<p>SirA is an essential gene in latent tuberculosis infections</p><br />
</div><br />
<div class="results"><br />
<h2>Results</h2><br />
<ul><br />
<li>Produced an <i>E. coli</i> strain which relies upon mycobacterial sirA, fprA and fdxA genes to survive in M9 minimal media</li><br />
<li>Demonstrated that <i>E. coli</i> can survive with mycobacterial sulfite reduction pathway with Flux Balance Analysis</li><br />
<li>Located drug target sites on sirA as well as identified high structural similarity between cysI and sirA through structural anaylsis</li><br />
</ul><br />
<p></p><br />
</div><br />
<div class="biocriks"><br />
<h2>BioBricks</h2><br />
<ol><br />
<li><a href="http://parts.igem.org/Part:BBa_K1137000">BBa_K1137000 (SirA)</a></li><br />
<li><a href="http://parts.igem.org/Part:BBa_K1137001">BBa_K1137001 (FprA)</a></li> <br />
<li><a href="http://parts.igem.org/Part:BBa_K1137002">BBa_K1137002 (FdxA)</a></li><br />
</ul><br />
</div><br />
<div style="clear: both;"></div><br />
<div class="aims"><br />
<h2>Aims</h2><br />
<p>To perform a drug screen targeted at the sirA gene from mycobacteria</p><br />
</div><br />
<a href="#Introduction"><br />
<div class="hlink"><br />
<h2>Skip to Introduction</h2><br />
</div><br />
</a><br />
<a href="#Model"><br />
<div class="hlink"><br />
<h2>Skip to Modeling</h2><br />
</div><br />
</a><br />
<a href="#Design"><br />
<div class="hlink"><br />
<h2>Skip to Design</h2><br />
</div><br />
</a><br />
<a href="#Results"> <br />
<div class="hlink" style="margin-right:0px"><br />
<h2>Skip to Results</h2><br />
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</div><br />
<div id="Introduction"></div><br />
<h2>Introduction</h2><br />
<div class="leftparagraph"><br />
<p> &nbsp;&nbsp;<br />
SirA is essential for <i>M. tuberculosis</i> persistence phenotype as sulfur containing amino acids are particularly sensitive to oxidative stress within the macrophage and must regularly be replaced <a href="#Reference">(Pinto <i>et al</i> 2007)</a>. Currently, there are no drug candidates that are known to specifically inhibit SirA and conventional drug screens involve do not provide information regarding the mechanism of drug action nor do compounds that inhibit exponential growth necessarily have an effect on persistent TB. We designed a working drug screen assay to specifically target the mycobacterial sulfite reductase protein SirA. To this end we cloned Ito <i>E. coli </i><span style="font-style: normal;">the sulfite reduction pathway</span> of <i>M. smegmatis</i>, a non-pathogenic mycobacterial relative of <i>M. Tuberculosis</i>. Our model overcomes the problem of long doubling time of <i>M. tuberculosis</i>. Specific inhibition of the sulfite reduction pathway is scored by comparing a drug screen of our <i>E. coli</i> construct <i>vs.</i> wild-type. Any drug candidates that have activity against both the wild-type <i>E. coli</i> and our construct are non-specific inhibitors of <i>E. coli</i> growth. However, any drug candidates that inhibit only the growth of our <i>E. coli </i>construct will be <span style="font-style: normal;">SirA</span><i> </i><span style="font-style: normal;">pathway specific.</span> <br />
</p><br />
</div><br />
<div class="rightparagraph"><br />
<a href="https://static.igem.org/mediawiki/2013/4/4f/PS_Drug_Scheme.png" target="_blank"><img src="https://static.igem.org/mediawiki/2013/4/4f/PS_Drug_Scheme.png" width="535px"/></a><br />
<p><b>Figure 1: Overview of Targeted Drug Screen Design</b></p><br />
</div><br />
<div id="Model"></div><br />
<div style="clear: both;"></div><br />
<h2>Flux Balance Analysis of Sulfite Reduction Pathway</h2><br />
<div class="leftparagraph"><br />
<p>&nbsp;&nbsp;<br />
We used an <i>E. coli</i> model (iJR904) obtained from the <a href="http://bigg.ucsd.edu/bigg/main.pl">BiGG database</a> as a starting model to obtain wild-type growth rate (f = 0.9129 divisions/hour). We then deleted the reaction ‘SULR’ which encodes for the sulphite reduction pathway involving cysI and obtained a f= -8e-13=0 divisions/hour indicating that the sulphite reduction pathway is essential for growth. Finally we introduced two new reactions for sirA and fprA and a new species fdxA. We found that growth with the mycobacteria pathway reverts the growth phenotype back to wild-type levels (f = 0.9105 divisions/hour). We then wanted to expand our model to find new pathways that we could utilize for a targeted drug screen approach. We wrote a matlab script that finds all the essential reactions in <i>M. tuberculosis</i> and all the essential reactions in <i>E. coli</i>, and then tries to complement the essential reactions in the <i>E. coli</i> model with the essential reactions from <i>M. tuberculosis</i>. The model identified <a href="https://2013.igem.org/Team:Paris_Bettencourt/Project/Target/FBA">100 metabolic reactions</a> that we could target. Additionally, due to the modular nature of the model, it can be used to find target-able metabolic reactions in any SBML file. The Matlab scripts can be found <a href="https://2013.igem.org/File:TargetFBA.zip">here</a> and requires <a href="http://opencobra.sourceforge.net/openCOBRA/Welcome.html">Cobra Toolbox 2.0</a> to function. Please visit the FBA page for a detailed list of <a href="https://2013.igem.org/Team:Paris_Bettencourt/Project/Target/FBA">results</a>.<br />
</p><br />
</div><br />
<div class="rightparagraph"><br />
<center><a href="https://static.igem.org/mediawiki/2013/7/76/PS_FBA.png" target="_blank"><img src="https://static.igem.org/mediawiki/2013/7/76/PS_FBA.png" width="267.5px"/></a></center><br />
<p><b>Figure 2: Biomass Flux through <i>E. coli</i> and mycoSIR E. coli</b><div style="font-size: 90%">Flux balance analysis was run using Cobra Toolbox 2.0 on <i>E. coli</i> sbml model iJR904 with and without SULR reaction. Additionally an <i>E. coli</i> sbml model was built with the SULR reaction replaced with a reaction representing the mycobacterial SirA reaction and FprA reaction, as well as ferredoxin FdxA as an additional species. The Biomass flux is restored to 99.75% of the wild-type level with the synthetic mycobacterial system.</div></p><br />
</div><br />
<div id="Model"></div><br />
<div style="clear: both;"></div><br />
<br />
<h2>Structural Analysis of SirA</h2><br />
<div class="leftparagraph"><br />
<p>&nbsp;&nbsp;<br />
Superimposing the structures of </span><i>M.tuberculosis</i><span style="font-style: normal;"> SirA and </span><i>E.coli </i><span style="font-style: normal;">CysI reveals high homology, in particular of the active sites. Both proteins have the same symmetry (psuedo 2 fold) indicative of a common evolutionary origin. Our analysis highlighted important conserved residues, involved in substrate binding to be Arg97, Arg130, Arg166, Lys207. These positively charged residues are conserved in the sulphite/nitrite reductase family. In addition, 4 Cys residues are conserved for iron-sulphur binding. </span><br />
<p>The most profound structural differences between the two enzymes are found in the ferredoxin binding site and SirA's most C terminal residues and several surface loop regions due to deletions or insertions. A stark difference is a covalent bond formed between Cys161 (thiolate) and Tyr69 (C carbon atom) found adjacent to the redox center (Cu ions) in SirA. The covalently bound residues act as a secondary cofactor in tyrosyl radical stabilization. </p> <br />
</p><br />
</div><br />
<div class="rightparagraph"><br />
<center><a href="https://static.igem.org/mediawiki/2013/5/52/Purplecys_SirA-1.png" target="_blank"><img src="https://static.igem.org/mediawiki/2013/5/52/Purplecys_SirA-1.png" width="267.5px"/></a></center><br />
<p><b>Figure 3: The superimposed 3D protein structures of SirA and CysI.</b><div style="font-size: 90%"> 303 amino acids are involved in superimposition with an rsmd of 1.41Å. All domains and loops of CysI are coloured purple, whilst SirA is coloured according to structural similarity with CysI: Red indicates poor alignment whilst blue indicates good alignment.</div></p><br />
</div><br />
<div id="Model"></div><br />
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<h2>Identification of potential drug target binding sites</h2><br />
<div class="leftparagraph"><br />
<p>&nbsp;&nbsp;<br />
Our structural analysis provided the basis for our drug target prediction. Using Chembl and swiss pdb, we have shown a predicted drug target site. Our calculation gives strong favour for a drug to be effective at this site. The calculation reflects the suitability of small molecules to the binding site under the Lipinski's Rule of 5.</p><br />
<p>The drug target is located at the interface of the three domains. This binding pocket exhibits a dense hydrophobic region. Our analysis targets 48 amino acids of SirA within 6Å of a modelled small drug molecule. Of these residues, only 6 amino acids are charged: His409, Asp453, Asp474, His500, Asp504 and Arg541.<br />
</p><br />
</div><br />
<div class="rightparagraph"><br />
<center><a href="https://static.igem.org/mediawiki/2013/5/59/Drug_target_withoutAA.png" target="_blank"><img src="https://static.igem.org/mediawiki/2013/5/59/Drug_target_withoutAA.png" width="267.5px"/></a></center><br />
<p><b>Figure 4 Drug target locations in SirA </b><div style="font-size: 90%">A domain located in SirA, identified as a drug target through Chembl analysis.</div></p><br />
</div><br />
<div style="clear: both;"></div><br />
<h2>Structure based pharmacophore modelling of mycobacterial Fpra</h2><br />
<div class="leftparagraph"><br />
<p>&nbsp;&nbsp;<br />
Using LigandScount 3.1, we searched over 8100 drug compounds from the BindingDB and Chembl databases for drugs targetting mycobacterial Fpra. Our search revealed Riboflavin (Vitamin B2) and Pyridoxine to be drug targets for Fpra. We used NADP interacting with the active site as the model of the pharmacore. Results showed pyridoxin to be a competitive inhibitor to NADP. Pyridoxin is a synthetic compound currently available as a prescribed drug. </span><br />
<p>Chembl analysis of Pyridoxine (vitamin B6) show that it's properties fulfill Lipinski's criteria of being an orally active drug in humans. These properties state that any small drug molecule must have: no more than 5 H bond donors, no more 10 H bond acceptors (N or O atoms), mol mass of less than 500 dalts and octanol-water partition coefficient log P of no greater than 5).</p> <br />
</p><br />
<p>We have shown the proposed properties of Pyridoxine's interaction with Fpra as a competitive inhibitor to NADP at Fpra's active site. The key amino acids at the active site are Ala205, GLN204 and Thr208. GLN204 and Ala205 act as hydrogen bond acceptors whilst Thr208 interacts with a H via van der waals forces. Pyridoxin is a smaller, more lipid soluble molecule than NADP, thus more fitting to Lipinski's criteria. </p> <br />
</p><br />
</div><br />
<div class="rightparagraph"><br />
<center><a href="https://static.igem.org/mediawiki/2013/6/60/Fnr_ribbons.png" target="_blank"><img src="https://static.igem.org/mediawiki/2013/5/52/Fnr_ribbons.png" width="267.5px"/></a></center><br />
<p><b>Figure 5: Our 3D model shows the structure of FNR where negative residues are coloured in blue, positive residues in red and NAD in purple (ball and stick representation). The key amino acids at the active site are Glu211, Gly 366, Arg 110, Arg 199, Arg 200 and Asn155. Glu211 acts as a hydrogen acceptor whilst the latter four residues act as hydrogen donors.</div></p><br />
</div><br />
<div id="Model"></div><br />
<div style="clear: both;"></div><br />
<p><b>Figure 6: Comparison of NADP interaction with Fpra's active site and Pyridoxine's interaction to it's active site. </div></p><br />
</div><br />
<div id="Model"></div><br />
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<div id="Design"></div><br />
<h2>Synthetic Mycobacteria Pathway</h2><br />
<div class="leftparagraph"><br />
<p> &nbsp;&nbsp;<br />
We designed a synthetic </span><i>M.smegmatis-</i><span style="font-style: normal;">derived sulfite reduction pathway containing sirA - the sulfite reductase, and two supporting genes that are required for its function in </span><i>E.coli</i><span style="font-style: normal;">: fdxA and fprA. FdxA is a mycobacterial Ferredoxin cofactor which is oxidised by SirA during the sulfite reduction reaction and FprA is a Ferredoxin-NADPH reductase use replenish the reduced Fdx pool. The genes' sequences were taken from previous work describing their expression <a href="#Reference">(Pinto <i>et al</i> 2007)</a> in </span><i>E.coli</i><span style="font-style: normal;"> for purification and in vitro characterization; we removed restriction sites and codon optimized for expression in </span><i>E. coli</i><span style="font-style: normal;">. The genes were then cloned into two Duet expression vectors, one containing sirA and one containing the supporting genesand were transformed into our knock-out mutant strains of </span><i>E. coli</i>. Data on Growth curves can be found <a href="https://2013.igem.org/Team:Paris_Bettencourt/Notebook/Drug_Screening/Monday_30th_September.html">here</a>. <br />
</p><br />
</div><br />
<div class="rightparagraph"><br />
<a href="https://static.igem.org/mediawiki/2013/8/84/TB_drug_FinalSmeg.png" target="_blank"><img src="https://static.igem.org/mediawiki/2013/8/84/TB_drug_FinalSmeg.png" width="535px"/></a><br />
<p><b>Figure 5: Growth curves of <i>E. coli</i> mycoSIR</b> <div style="font-size: 90%">BL21 (DE3) ΔcysI containing the MycoSIR pathway (MycoSIR <i>E. coli</i>) were grown in liquid minimal media containing Various concentration of IPTG. (A) Replicates of each strain were measured for absorbance in a spectrophotometer every 10 minutes for 14 hours. Growth was observed for the WT BL21 <i>E. coli</i>, (blue), and the MycoSIR <i>E. coli</i> (red). No growth was detected for uninduced MycoSIR <i>E. coli</i> (purple) or for the BL21 (DE3) ΔcysI that did not contain the synthetic pathway (Orange) . (B) Mean Final ODs of all replicates, measured after 14 hours of growth. Growth was detected in zmSIR <i>E. coli</i> and WT BL21 but not in uninduced zmSIR strain.</div></p> <br />
</div><br />
<div style="clear: both;"></div><br />
<h2>Creation of Knock out Mutants</h2><br />
<div class="leftparagraph"><br />
<p>&nbsp;&nbsp;<br />
We prepared two strains of <i>E. coli</i> which have the sulfite reduction pathway deleted: BL21 (DE3) <i>ΔCysI Δfpr ΔydbK</i> and BL21 (AI) <i>ΔCysI</i>. CysI is responsible for sulfite reduction in <i>E. coli</i>, while <i>fpr and ydbK</i> are two non-essential genes that consume ferredoxin. These two genes are deleted, as sulfite reduction in mycobacteria is ferredoxin dependent in comparison to<i> E. coli</i> in which it is NADPH dependant. These genes were also removed to ensure that they do not interfere with our system. <br />
</div><br />
<div class="rightparagraph"><br />
</div><br />
<div style="clear: both;"></div><br />
<h2>Synthetic Corn Pathway</h2><br />
<div class="leftparagraph"><br />
<p>&nbsp;&nbsp;<br />
Additionally we prototyped the system with a reconstruction of a sulphite reduction pathway previously designed and published by the silver group <a href="#Reference">(2011 Barstow et al)</a>. In place of CysI, a corn (Zea mays) derived sulfite reductase (zmSIR) was used. Two additional genes were included: Spinach ferredoxin (soFD), and corn derived ferredoxin NADP+ reductase (zmFNR). These genes, respectively, are required for production of the ferredoxin cofactor and the NADP+ ferredoxin reductase and are required for sulfite reductase (zmSIR) to function within <i>E. coli</i>.<br />
</p><br />
</div><br />
<div class="rightparagraph"><br />
<a href="https://static.igem.org/mediawiki/2013/a/a2/PB_final_Corn.png" target="_blank"><img src="https://static.igem.org/mediawiki/2013/a/a2/PB_final_Corn.png" width="535px"/></a><br />
<p><b>Figure 6: Growth curves of <i>E. coli</i> maizeSIR</b><div style="font-size: 90%"> BL21 (DE3) ΔcysI containing the MaizeSIR pathway (MaizeSIR <i>E. coli</i>) were grown in liquid minimal media containing Various concentration of IPTG. (A) Replicates of each strain were measured for absorbance in a spectrophotometer every 10 minutes for 14 hours. Growth was observed for the WT BL21 <i>E. coli</i>, (blue), and the MaizeSIR <i>E. coli</i> (red). No growth was detected for uninduced MaizeSIR <i>E. coli</i> (purple) or for the BL21 (DE3) ΔcysI that did not contain the synthetic pathway (Orange) . (B) Mean Final ODs of all replicates, measured after 14 hours of growth. Growth was detected in zmSIR <i>E. coli</i> and WT BL21 but not in uninduced zmSIR strain.</div></p> <br />
</div><br />
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<div id="Results"></div><br />
<h2>Results</h2><br />
<div class="leftparagraph"><br />
<p>&nbsp;&nbsp;<br />
Upon successful cloning of the three genes into our <i>E. coli</i> deletion strain, we continued to confirm that all three genes are required for growth on minimal media. Our two synthetic pathways were found to rescue growth on a sulfurless amino acid supplemented minimal media. We hope that this technique of using synthetic biology to overcome problems faced in naturally occurring systems will be both a large boon to the pursuit of finding novel drug candidates in <i>M. tuberculosis</i> and more broadly as this technique can be used for high-throughput screening of any pathway that can be constructed to be essential for growth in <i>E. coli</i>.<br />
</p><br />
</div><br />
<div class="rightparagraph"><br />
<a href="https://static.igem.org/mediawiki/2013/8/85/PS_D_Figure1_plate.png" target="_blank"><img src="https://static.igem.org/mediawiki/2013/8/85/PS_D_Figure1_plate.png" width="535px"/></a><br />
<p><b>Figure 7: Growth of zmSIR <i>E. coli</i> on minimal media.</b> <div style="font-size: 90%">BL21 (DE3) ΔcysI cells transformed with 1, 2 and 3 genes of the 3-gene zmSIR synthetic pathway were grown for 24 hours on minimal media supplemented with 25 uM IPTG (see methods), along with a WT BL21 (DE3) serving as a negative control, and an untransformed BL21 (DE3) ΔcysI, as negative control. Rescue of growth required all genes of the synthetic pathway (SIR, FNR and FD). </div></p><br />
</div><br />
<div style="clear: both;"></div><br />
<br />
<h2>Z-score</h2><br />
<div class="leftparagraph"><br />
<p>&nbsp;&nbsp;<br />
The Z-score is a statistical measurement aimed at assessing the "hit effect" in a drug screen high throughput screening. It is a commonly used measurement that shows how well did the drug effect the growth of the assay strain and how significant is the decrease in growth.</p><br />
<br />
<p>&nbsp;&nbsp;<br />
To calculate the Z-score we used our experimental <i>E. coli</i> strain BL21 (AI) ΔcysI that carries all three genes of the synthetic pathway (sirA, fprA, fdxA). We grew it in the M9 minimal media supplemented with amino acid sulfur dropout powder, in a 96 well plate. Four of the wells were "spiked" with antibiotics (Amp, Gent, Kan, and Spect). </p><br />
</div><br />
<div class="rightparagraph"><br />
<p>&nbsp;&nbsp;<br />
This is basically a simulation of the drug screen but without the actual drugs. Only the drug screen controls are used: growth in M9 as a negative control (no drugs) and growth in M9 + antibiotics as a positive control (a sure hit). Then we compared the distribution of the growth in the negative control with the distribution of growth in the positive control. The Z-score shows how many standard deviations away from each other are the means of the two distributions.</p><br />
<p><b>Our Z-score is: -10.2.</b></div></p><br />
<br />
<div style="clear: both;"></div><br />
<br />
<h2>Z-factor</h2><br />
<div class="leftparagraph"><br />
<p>&nbsp;&nbsp;<br />
Z-factor is a measurement complementary to the Z-score. It measures the assay's quality based on the same data extracted from the same experiment made for the Z-score. This calculation gives an estimation of how far the negative controls are from the positive controls. It is a comparison of the two distributions which assumes that both distributions are normal and calculate how far 99% of the data points of each distribution are from each other.</p><br />
<br />
</div><br />
<div class="rightparagraph"><br />
<p>&nbsp;&nbsp;<br />
Z-factor is given on a scale from 0 to 1. Scores between 0.5 and 1 show that the assay is good and will enable testing in High throughput screens.</p><br />
<p><b>Our Z-factor score is 0.58.</b></div></p><br />
</div><br />
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<div id="Reference"></div><br />
<h2>Literature</h2><br />
<div class="leftparagraph"><br />
<ul><br />
<li>Global Alliance for TB Drug Development, Tuberculosis. Scientific blueprint for tuberculosis drug development, Tuberculosis (Edinb) 81 Suppl 1, 1–52 (2001).</li><br />
<br />
<li>World Health Organization, Global Tuberculosis Report 2012 (2012).</li><br />
<br />
<li>K. Raman, K. Yeturu, N. Chandra, targetTB: A target identification pipeline for Mycobacterium tuberculosis through an interactome, reactome and genome-scale structural analysis, BMC Syst Biol 2, 109 (2008).</li><br />
<br />
<li>R. Pinto, J. S. Harrison, T. Hsu, W. R. Jacobs, T. S. Leyh, Sulfite Reduction in Mycobacteria, Journal of Bacteriology 189, 6714–6722 (2007).</li><br />
<br />
<li>B. Barstow C. M. Agapakis, P. M. Boyle, G. Grandl, P. A. Silver, E. H. Wintermute, A synthetic system links FeFe-hydrogenases to essential E. coli sulfur metabolism, J Biol Eng 5, 7 (2011).</li><br />
</ul><br />
<br />
</div><br />
</div><br />
<div class="rightparagraph"><br />
<ul><br />
<li>Schellenberger J, Que R, Fleming RMT, Thiele I, Orth JD, Feist AM, Zielinski DC, Bordbar A, Lewis NE, Rahmanian S, Kang J, Hyduke DR, Palsson BØ. 2011 Quantitative prediction of cellular metabolism with constraint-based models: the COBRA Toolbox v2.0. Nature Protocols 6:1290-1307.</li><br />
<br />
<li>Schellenberger, J., Park, J. O., Conrad, T. C., and Palsson, B. Ø., BiGG: a Biochemical Genetic and Genomic knowledgebase of large scale metabolic reconstructions, BMC Bioinformatics, 11:213, (2010).</li><br />
<br />
<li>S. G. Franzblau et al., Comprehensive analysis of methods used for the evaluation of compounds against Mycobacterium tuberculosis, Tuberculosis 92, 453–488 (2012).</li><br />
<br />
<li>D. J. Payne, M. N. Gwynn, D. J. Holmes, D. L. Pompliano, Drugs for bad bugs: confronting the challenges of antibacterial discovery, Nat Rev Drug Discov 6, 29–40 (2006).</li><br />
<br />
<li>M. Nakayama, T. Akashi, T. Hase, Plant sulfite reductase: molecular structure, catalytic function and interaction with ferredoxin, J. Inorg. Biochem. 82, 27–32 (2000).</li><br />
</ul><br />
<br />
</div><br />
<div style="clear: both;"></div><br />
<h2>Attributions</h2><br />
<p>Strains NEBTurbo, BL21 (DE3) KO20, BL21 AI were provided by INSERM U1001.</p><br />
<p>Plasmids pET Duet, pACYC Duet, pACYC zmSIR, pACYC soFD zmSIR, pCDF FNR were provided by INSERM U1001.</p><br />
<p>Genes msSirA, msFprA, msFdxA were synthesized by IDT.</p><br />
<p>Project was designed by Idonnya Aghoghogbe, Yonatan Zegman, Matthew Deyell and Edwin Wintermute. All experiments and modelling were performed by Idonnya Aghoghogbe, Yonatan Zegman, Matthew Deyell. </p><br />
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{{:Team:Paris_Bettencourt/footer}}</div>Idonnyahttp://2013.igem.org/Team:Paris_Bettencourt/Project/TargetTeam:Paris Bettencourt/Project/Target2013-10-29T01:50:16Z<p>Idonnya: </p>
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<div style="width:1100px;margin:0 auto;"><br />
<img src="https://static.igem.org/mediawiki/2013/3/3a/PB_logoParis.gif" width="122px" style="position:absolute;top:40px;right:30px;"/><br />
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<img src="https://static.igem.org/mediawiki/2013/c/c7/PB_targettitle.png" style="margin-bottom:15px"/><br />
<div class="overbox"><br />
<div class="bkgr"><br />
<h2>Background</h2><br />
<p>SirA is an essential gene in latent tuberculosis infections</p><br />
</div><br />
<div class="results"><br />
<h2>Results</h2><br />
<ul><br />
<li>Produced an <i>E. coli</i> strain which relies upon mycobacterial sirA, fprA and fdxA genes to survive in M9 minimal media</li><br />
<li>Demonstrated that <i>E. coli</i> can survive with mycobacterial sulfite reduction pathway with Flux Balance Analysis</li><br />
<li>Located drug target sites on sirA as well as identified high structural similarity between cysI and sirA through structural anaylsis</li><br />
</ul><br />
<p></p><br />
</div><br />
<div class="biocriks"><br />
<h2>BioBricks</h2><br />
<ol><br />
<li><a href="http://parts.igem.org/Part:BBa_K1137000">BBa_K1137000 (SirA)</a></li><br />
<li><a href="http://parts.igem.org/Part:BBa_K1137001">BBa_K1137001 (FprA)</a></li> <br />
<li><a href="http://parts.igem.org/Part:BBa_K1137002">BBa_K1137002 (FdxA)</a></li><br />
</ul><br />
</div><br />
<div style="clear: both;"></div><br />
<div class="aims"><br />
<h2>Aims</h2><br />
<p>To perform a drug screen targeted at the sirA gene from mycobacteria</p><br />
</div><br />
<a href="#Introduction"><br />
<div class="hlink"><br />
<h2>Skip to Introduction</h2><br />
</div><br />
</a><br />
<a href="#Model"><br />
<div class="hlink"><br />
<h2>Skip to Modeling</h2><br />
</div><br />
</a><br />
<a href="#Design"><br />
<div class="hlink"><br />
<h2>Skip to Design</h2><br />
</div><br />
</a><br />
<a href="#Results"> <br />
<div class="hlink" style="margin-right:0px"><br />
<h2>Skip to Results</h2><br />
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</div><br />
<div id="Introduction"></div><br />
<h2>Introduction</h2><br />
<div class="leftparagraph"><br />
<p> &nbsp;&nbsp;<br />
SirA is essential for <i>M. tuberculosis</i> persistence phenotype as sulfur containing amino acids are particularly sensitive to oxidative stress within the macrophage and must regularly be replaced <a href="#Reference">(Pinto <i>et al</i> 2007)</a>. Currently, there are no drug candidates that are known to specifically inhibit SirA and conventional drug screens involve do not provide information regarding the mechanism of drug action nor do compounds that inhibit exponential growth necessarily have an effect on persistent TB. We designed a working drug screen assay to specifically target the mycobacterial sulfite reductase protein SirA. To this end we cloned Ito <i>E. coli </i><span style="font-style: normal;">the sulfite reduction pathway</span> of <i>M. smegmatis</i>, a non-pathogenic mycobacterial relative of <i>M. Tuberculosis</i>. Our model overcomes the problem of long doubling time of <i>M. tuberculosis</i>. Specific inhibition of the sulfite reduction pathway is scored by comparing a drug screen of our <i>E. coli</i> construct <i>vs.</i> wild-type. Any drug candidates that have activity against both the wild-type <i>E. coli</i> and our construct are non-specific inhibitors of <i>E. coli</i> growth. However, any drug candidates that inhibit only the growth of our <i>E. coli </i>construct will be <span style="font-style: normal;">SirA</span><i> </i><span style="font-style: normal;">pathway specific.</span> <br />
</p><br />
</div><br />
<div class="rightparagraph"><br />
<a href="https://static.igem.org/mediawiki/2013/4/4f/PS_Drug_Scheme.png" target="_blank"><img src="https://static.igem.org/mediawiki/2013/4/4f/PS_Drug_Scheme.png" width="535px"/></a><br />
<p><b>Figure 1: Overview of Targeted Drug Screen Design</b></p><br />
</div><br />
<div id="Model"></div><br />
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<h2>Flux Balance Analysis of Sulfite Reduction Pathway</h2><br />
<div class="leftparagraph"><br />
<p>&nbsp;&nbsp;<br />
We used an <i>E. coli</i> model (iJR904) obtained from the <a href="http://bigg.ucsd.edu/bigg/main.pl">BiGG database</a> as a starting model to obtain wild-type growth rate (f = 0.9129 divisions/hour). We then deleted the reaction ‘SULR’ which encodes for the sulphite reduction pathway involving cysI and obtained a f= -8e-13=0 divisions/hour indicating that the sulphite reduction pathway is essential for growth. Finally we introduced two new reactions for sirA and fprA and a new species fdxA. We found that growth with the mycobacteria pathway reverts the growth phenotype back to wild-type levels (f = 0.9105 divisions/hour). We then wanted to expand our model to find new pathways that we could utilize for a targeted drug screen approach. We wrote a matlab script that finds all the essential reactions in <i>M. tuberculosis</i> and all the essential reactions in <i>E. coli</i>, and then tries to complement the essential reactions in the <i>E. coli</i> model with the essential reactions from <i>M. tuberculosis</i>. The model identified <a href="https://2013.igem.org/Team:Paris_Bettencourt/Project/Target/FBA">100 metabolic reactions</a> that we could target. Additionally, due to the modular nature of the model, it can be used to find target-able metabolic reactions in any SBML file. The Matlab scripts can be found <a href="https://2013.igem.org/File:TargetFBA.zip">here</a> and requires <a href="http://opencobra.sourceforge.net/openCOBRA/Welcome.html">Cobra Toolbox 2.0</a> to function. Please visit the FBA page for a detailed list of <a href="https://2013.igem.org/Team:Paris_Bettencourt/Project/Target/FBA">results</a>.<br />
</p><br />
</div><br />
<div class="rightparagraph"><br />
<center><a href="https://static.igem.org/mediawiki/2013/7/76/PS_FBA.png" target="_blank"><img src="https://static.igem.org/mediawiki/2013/7/76/PS_FBA.png" width="267.5px"/></a></center><br />
<p><b>Figure 2: Biomass Flux through <i>E. coli</i> and mycoSIR E. coli</b><div style="font-size: 90%">Flux balance analysis was run using Cobra Toolbox 2.0 on <i>E. coli</i> sbml model iJR904 with and without SULR reaction. Additionally an <i>E. coli</i> sbml model was built with the SULR reaction replaced with a reaction representing the mycobacterial SirA reaction and FprA reaction, as well as ferredoxin FdxA as an additional species. The Biomass flux is restored to 99.75% of the wild-type level with the synthetic mycobacterial system.</div></p><br />
</div><br />
<div id="Model"></div><br />
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<br />
<h2>Structural Analysis of SirA</h2><br />
<div class="leftparagraph"><br />
<p>&nbsp;&nbsp;<br />
Superimposing the structures of </span><i>M.tuberculosis</i><span style="font-style: normal;"> SirA and </span><i>E.coli </i><span style="font-style: normal;">CysI reveals high homology, in particular of the active sites. Both proteins have the same symmetry (psuedo 2 fold) indicative of a common evolutionary origin. Our analysis highlighted important conserved residues, involved in substrate binding to be Arg97, Arg130, Arg166, Lys207. These positively charged residues are conserved in the sulphite/nitrite reductase family. In addition, 4 Cys residues are conserved for iron-sulphur binding. </span><br />
<p>The most profound structural differences between the two enzymes are found in the ferredoxin binding site and SirA's most C terminal residues and several surface loop regions due to deletions or insertions. A stark difference is a covalent bond formed between Cys161 (thiolate) and Tyr69 (C carbon atom) found adjacent to the redox center (Cu ions) in SirA. The covalently bound residues act as a secondary cofactor in tyrosyl radical stabilization. </p> <br />
</p><br />
</div><br />
<div class="rightparagraph"><br />
<center><a href="https://static.igem.org/mediawiki/2013/5/52/Purplecys_SirA-1.png" target="_blank"><img src="https://static.igem.org/mediawiki/2013/5/52/Purplecys_SirA-1.png" width="267.5px"/></a></center><br />
<p><b>Figure 3: The superimposed 3D protein structures of SirA and CysI.</b><div style="font-size: 90%"> 303 amino acids are involved in superimposition with an rsmd of 1.41Å. All domains and loops of CysI are coloured purple, whilst SirA is coloured according to structural similarity with CysI: Red indicates poor alignment whilst blue indicates good alignment.</div></p><br />
</div><br />
<div id="Model"></div><br />
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<h2>Identification of potential drug target binding sites</h2><br />
<div class="leftparagraph"><br />
<p>&nbsp;&nbsp;<br />
Our structural analysis provided the basis for our drug target prediction. Using Chembl and swiss pdb, we have shown a predicted drug target site. Our calculation gives strong favour for a drug to be effective at this site. The calculation reflects the suitability of small molecules to the binding site under the Lipinski's Rule of 5.</p><br />
<p>The drug target is located at the interface of the three domains. This binding pocket exhibits a dense hydrophobic region. Our analysis targets 48 amino acids of SirA within 6Å of a modelled small drug molecule. Of these residues, only 6 amino acids are charged: His409, Asp453, Asp474, His500, Asp504 and Arg541.<br />
</p><br />
</div><br />
<div class="rightparagraph"><br />
<center><a href="https://static.igem.org/mediawiki/2013/5/59/Drug_target_withoutAA.png" target="_blank"><img src="https://static.igem.org/mediawiki/2013/5/59/Drug_target_withoutAA.png" width="267.5px"/></a></center><br />
<p><b>Figure 4 Drug target locations in SirA </b><div style="font-size: 90%">A domain located in SirA, identified as a drug target through Chembl analysis.</div></p><br />
</div><br />
<div style="clear: both;"></div><br />
<h2>Structure based pharmacophore modelling of mycobacterial Fpra</h2><br />
<div class="leftparagraph"><br />
<p>&nbsp;&nbsp;<br />
Using LigandScount 3.1, we searched over 8100 drug compounds from the BindingDB and Chembl databases for drugs targetting mycobacterial Fpra. Our search revealed Riboflavin (Vitamin B2) and Pyridoxine to be drug targets for Fpra. We used NADP interacting with the active site as the model of the pharmacore. Results showed pyridoxin to be a competitive inhibitor to NADP. Pyridoxin is a synthetic compound currently available as a prescribed drug. </span><br />
<p>Chembl analysis of Pyridoxine (vitamin B6) show that it's properties fulfill Lipinski's criteria of being an orally active drug in humans. These properties state that any small drug molecule must have: no more than 5 H bond donors, no more 10 H bond acceptors (N or O atoms), mol mass of less than 500 dalts and octanol-water partition coefficient log P of no greater than 5).</p> <br />
</p><br />
<p>We have shown the proposed properties of Pyridoxine's interaction with Fpra as a competitive inhibitor to NADP at Fpra's active site. The key amino acids at the active site are Ala205, GLN204 and Thr208. GLN204 and Ala205 act as hydrogen bond acceptors whilst Thr208 interacts with a H via van der waals forces. Pyridoxin is a smaller, more lipid soluble molecule than NADP, thus more fitting to Lipinski's criteria. </p> <br />
</p><br />
</div><br />
<div class="rightparagraph"><br />
<center><a href="https://static.igem.org/mediawiki/2013/6/60/Fnr_ribbons.png" target="_blank"><img src="https://static.igem.org/mediawiki/2013/5/52/Fnr_ribbons.png" width="267.5px"/></a></center><br />
<p><b>Figure 5: Our 3D model shows the structure of FNR where negative residues are coloured in blue, positive residues in red and NAD in purple (ball and stick representation). The key amino acids at the active site are Glu211, Gly 366, Arg 110, Arg 199, Arg 200 and Asn155. Glu211 acts as a hydrogen acceptor whilst the latter four residues act as hydrogen donors.</div></p><br />
</div><br />
<div id="Model"></div><br />
<div style="clear: both;"></div><br />
<p><b>Figure 6:Comparison of NADP interaction with Fpra's active site and Pyridoxine's interaction to it's active site. </div></p><br />
</div><br />
<div id="Model"></div><br />
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<div id="Design"></div><br />
<h2>Synthetic Mycobacteria Pathway</h2><br />
<div class="leftparagraph"><br />
<p> &nbsp;&nbsp;<br />
We designed a synthetic </span><i>M.smegmatis-</i><span style="font-style: normal;">derived sulfite reduction pathway containing sirA - the sulfite reductase, and two supporting genes that are required for its function in </span><i>E.coli</i><span style="font-style: normal;">: fdxA and fprA. FdxA is a mycobacterial Ferredoxin cofactor which is oxidised by SirA during the sulfite reduction reaction and FprA is a Ferredoxin-NADPH reductase use replenish the reduced Fdx pool. The genes' sequences were taken from previous work describing their expression <a href="#Reference">(Pinto <i>et al</i> 2007)</a> in </span><i>E.coli</i><span style="font-style: normal;"> for purification and in vitro characterization; we removed restriction sites and codon optimized for expression in </span><i>E. coli</i><span style="font-style: normal;">. The genes were then cloned into two Duet expression vectors, one containing sirA and one containing the supporting genesand were transformed into our knock-out mutant strains of </span><i>E. coli</i>. Data on Growth curves can be found <a href="https://2013.igem.org/Team:Paris_Bettencourt/Notebook/Drug_Screening/Monday_30th_September.html">here</a>. <br />
</p><br />
</div><br />
<div class="rightparagraph"><br />
<a href="https://static.igem.org/mediawiki/2013/8/84/TB_drug_FinalSmeg.png" target="_blank"><img src="https://static.igem.org/mediawiki/2013/8/84/TB_drug_FinalSmeg.png" width="535px"/></a><br />
<p><b>Figure 5: Growth curves of <i>E. coli</i> mycoSIR</b> <div style="font-size: 90%">BL21 (DE3) ΔcysI containing the MycoSIR pathway (MycoSIR <i>E. coli</i>) were grown in liquid minimal media containing Various concentration of IPTG. (A) Replicates of each strain were measured for absorbance in a spectrophotometer every 10 minutes for 14 hours. Growth was observed for the WT BL21 <i>E. coli</i>, (blue), and the MycoSIR <i>E. coli</i> (red). No growth was detected for uninduced MycoSIR <i>E. coli</i> (purple) or for the BL21 (DE3) ΔcysI that did not contain the synthetic pathway (Orange) . (B) Mean Final ODs of all replicates, measured after 14 hours of growth. Growth was detected in zmSIR <i>E. coli</i> and WT BL21 but not in uninduced zmSIR strain.</div></p> <br />
</div><br />
<div style="clear: both;"></div><br />
<h2>Creation of Knock out Mutants</h2><br />
<div class="leftparagraph"><br />
<p>&nbsp;&nbsp;<br />
We prepared two strains of <i>E. coli</i> which have the sulfite reduction pathway deleted: BL21 (DE3) <i>ΔCysI Δfpr ΔydbK</i> and BL21 (AI) <i>ΔCysI</i>. CysI is responsible for sulfite reduction in <i>E. coli</i>, while <i>fpr and ydbK</i> are two non-essential genes that consume ferredoxin. These two genes are deleted, as sulfite reduction in mycobacteria is ferredoxin dependent in comparison to<i> E. coli</i> in which it is NADPH dependant. These genes were also removed to ensure that they do not interfere with our system. <br />
</div><br />
<div class="rightparagraph"><br />
</div><br />
<div style="clear: both;"></div><br />
<h2>Synthetic Corn Pathway</h2><br />
<div class="leftparagraph"><br />
<p>&nbsp;&nbsp;<br />
Additionally we prototyped the system with a reconstruction of a sulphite reduction pathway previously designed and published by the silver group <a href="#Reference">(2011 Barstow et al)</a>. In place of CysI, a corn (Zea mays) derived sulfite reductase (zmSIR) was used. Two additional genes were included: Spinach ferredoxin (soFD), and corn derived ferredoxin NADP+ reductase (zmFNR). These genes, respectively, are required for production of the ferredoxin cofactor and the NADP+ ferredoxin reductase and are required for sulfite reductase (zmSIR) to function within <i>E. coli</i>.<br />
</p><br />
</div><br />
<div class="rightparagraph"><br />
<a href="https://static.igem.org/mediawiki/2013/a/a2/PB_final_Corn.png" target="_blank"><img src="https://static.igem.org/mediawiki/2013/a/a2/PB_final_Corn.png" width="535px"/></a><br />
<p><b>Figure 6: Growth curves of <i>E. coli</i> maizeSIR</b><div style="font-size: 90%"> BL21 (DE3) ΔcysI containing the MaizeSIR pathway (MaizeSIR <i>E. coli</i>) were grown in liquid minimal media containing Various concentration of IPTG. (A) Replicates of each strain were measured for absorbance in a spectrophotometer every 10 minutes for 14 hours. Growth was observed for the WT BL21 <i>E. coli</i>, (blue), and the MaizeSIR <i>E. coli</i> (red). No growth was detected for uninduced MaizeSIR <i>E. coli</i> (purple) or for the BL21 (DE3) ΔcysI that did not contain the synthetic pathway (Orange) . (B) Mean Final ODs of all replicates, measured after 14 hours of growth. Growth was detected in zmSIR <i>E. coli</i> and WT BL21 but not in uninduced zmSIR strain.</div></p> <br />
</div><br />
<div style="clear: both;"></div><br />
<div id="Results"></div><br />
<h2>Results</h2><br />
<div class="leftparagraph"><br />
<p>&nbsp;&nbsp;<br />
Upon successful cloning of the three genes into our <i>E. coli</i> deletion strain, we continued to confirm that all three genes are required for growth on minimal media. Our two synthetic pathways were found to rescue growth on a sulfurless amino acid supplemented minimal media. We hope that this technique of using synthetic biology to overcome problems faced in naturally occurring systems will be both a large boon to the pursuit of finding novel drug candidates in <i>M. tuberculosis</i> and more broadly as this technique can be used for high-throughput screening of any pathway that can be constructed to be essential for growth in <i>E. coli</i>.<br />
</p><br />
</div><br />
<div class="rightparagraph"><br />
<a href="https://static.igem.org/mediawiki/2013/8/85/PS_D_Figure1_plate.png" target="_blank"><img src="https://static.igem.org/mediawiki/2013/8/85/PS_D_Figure1_plate.png" width="535px"/></a><br />
<p><b>Figure 7: Growth of zmSIR <i>E. coli</i> on minimal media.</b> <div style="font-size: 90%">BL21 (DE3) ΔcysI cells transformed with 1, 2 and 3 genes of the 3-gene zmSIR synthetic pathway were grown for 24 hours on minimal media supplemented with 25 uM IPTG (see methods), along with a WT BL21 (DE3) serving as a negative control, and an untransformed BL21 (DE3) ΔcysI, as negative control. Rescue of growth required all genes of the synthetic pathway (SIR, FNR and FD). </div></p><br />
</div><br />
<div style="clear: both;"></div><br />
<br />
<h2>Z-score</h2><br />
<div class="leftparagraph"><br />
<p>&nbsp;&nbsp;<br />
The Z-score is a statistical measurement aimed at assessing the "hit effect" in a drug screen high throughput screening. It is a commonly used measurement that shows how well did the drug effect the growth of the assay strain and how significant is the decrease in growth.</p><br />
<br />
<p>&nbsp;&nbsp;<br />
To calculate the Z-score we used our experimental <i>E. coli</i> strain BL21 (AI) ΔcysI that carries all three genes of the synthetic pathway (sirA, fprA, fdxA). We grew it in the M9 minimal media supplemented with amino acid sulfur dropout powder, in a 96 well plate. Four of the wells were "spiked" with antibiotics (Amp, Gent, Kan, and Spect). </p><br />
</div><br />
<div class="rightparagraph"><br />
<p>&nbsp;&nbsp;<br />
This is basically a simulation of the drug screen but without the actual drugs. Only the drug screen controls are used: growth in M9 as a negative control (no drugs) and growth in M9 + antibiotics as a positive control (a sure hit). Then we compared the distribution of the growth in the negative control with the distribution of growth in the positive control. The Z-score shows how many standard deviations away from each other are the means of the two distributions.</p><br />
<p><b>Our Z-score is: -10.2.</b></div></p><br />
<br />
<div style="clear: both;"></div><br />
<br />
<h2>Z-factor</h2><br />
<div class="leftparagraph"><br />
<p>&nbsp;&nbsp;<br />
Z-factor is a measurement complementary to the Z-score. It measures the assay's quality based on the same data extracted from the same experiment made for the Z-score. This calculation gives an estimation of how far the negative controls are from the positive controls. It is a comparison of the two distributions which assumes that both distributions are normal and calculate how far 99% of the data points of each distribution are from each other.</p><br />
<br />
</div><br />
<div class="rightparagraph"><br />
<p>&nbsp;&nbsp;<br />
Z-factor is given on a scale from 0 to 1. Scores between 0.5 and 1 show that the assay is good and will enable testing in High throughput screens.</p><br />
<p><b>Our Z-factor score is 0.58.</b></div></p><br />
</div><br />
<div style="clear: both;"></div><br />
<div id="Reference"></div><br />
<h2>Literature</h2><br />
<div class="leftparagraph"><br />
<ul><br />
<li>Global Alliance for TB Drug Development, Tuberculosis. Scientific blueprint for tuberculosis drug development, Tuberculosis (Edinb) 81 Suppl 1, 1–52 (2001).</li><br />
<br />
<li>World Health Organization, Global Tuberculosis Report 2012 (2012).</li><br />
<br />
<li>K. Raman, K. Yeturu, N. Chandra, targetTB: A target identification pipeline for Mycobacterium tuberculosis through an interactome, reactome and genome-scale structural analysis, BMC Syst Biol 2, 109 (2008).</li><br />
<br />
<li>R. Pinto, J. S. Harrison, T. Hsu, W. R. Jacobs, T. S. Leyh, Sulfite Reduction in Mycobacteria, Journal of Bacteriology 189, 6714–6722 (2007).</li><br />
<br />
<li>B. Barstow C. M. Agapakis, P. M. Boyle, G. Grandl, P. A. Silver, E. H. Wintermute, A synthetic system links FeFe-hydrogenases to essential E. coli sulfur metabolism, J Biol Eng 5, 7 (2011).</li><br />
</ul><br />
<br />
</div><br />
</div><br />
<div class="rightparagraph"><br />
<ul><br />
<li>Schellenberger J, Que R, Fleming RMT, Thiele I, Orth JD, Feist AM, Zielinski DC, Bordbar A, Lewis NE, Rahmanian S, Kang J, Hyduke DR, Palsson BØ. 2011 Quantitative prediction of cellular metabolism with constraint-based models: the COBRA Toolbox v2.0. Nature Protocols 6:1290-1307.</li><br />
<br />
<li>Schellenberger, J., Park, J. O., Conrad, T. C., and Palsson, B. Ø., BiGG: a Biochemical Genetic and Genomic knowledgebase of large scale metabolic reconstructions, BMC Bioinformatics, 11:213, (2010).</li><br />
<br />
<li>S. G. Franzblau et al., Comprehensive analysis of methods used for the evaluation of compounds against Mycobacterium tuberculosis, Tuberculosis 92, 453–488 (2012).</li><br />
<br />
<li>D. J. Payne, M. N. Gwynn, D. J. Holmes, D. L. Pompliano, Drugs for bad bugs: confronting the challenges of antibacterial discovery, Nat Rev Drug Discov 6, 29–40 (2006).</li><br />
<br />
<li>M. Nakayama, T. Akashi, T. Hase, Plant sulfite reductase: molecular structure, catalytic function and interaction with ferredoxin, J. Inorg. Biochem. 82, 27–32 (2000).</li><br />
</ul><br />
<br />
</div><br />
<div style="clear: both;"></div><br />
<h2>Attributions</h2><br />
<p>Strains NEBTurbo, BL21 (DE3) KO20, BL21 AI were provided by INSERM U1001.</p><br />
<p>Plasmids pET Duet, pACYC Duet, pACYC zmSIR, pACYC soFD zmSIR, pCDF FNR were provided by INSERM U1001.</p><br />
<p>Genes msSirA, msFprA, msFdxA were synthesized by IDT.</p><br />
<p>Project was designed by Idonnya Aghoghogbe, Yonatan Zegman, Matthew Deyell and Edwin Wintermute. All experiments and modelling were performed by Idonnya Aghoghogbe, Yonatan Zegman, Matthew Deyell. </p><br />
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{{:Team:Paris_Bettencourt/footer}}</div>Idonnyahttp://2013.igem.org/File:Fnr_ribbons.pngFile:Fnr ribbons.png2013-10-29T01:25:57Z<p>Idonnya: Our 3D model shows the structure of FNR where negative residues are coloured in blue, positive residues in red and NAD in purple (ball and stick representation). The key amino acids at the active site are Glu211, Gly 366, Arg 110, Arg 199, Arg 200 and As</p>
<hr />
<div>Our 3D model shows the structure of FNR where negative residues are coloured in blue, positive residues in red and NAD in purple (ball and stick representation). The key amino acids at the active site are Glu211, Gly 366, Arg 110, Arg 199, Arg 200 and Asn155. Glu211 acts as a hydrogen acceptor whilst the latter four residues act as hydrogen donors.</div>Idonnyahttp://2013.igem.org/Team:Paris_Bettencourt/Notebook/Drug_Screening/Tuesday_6th_August.htmlTeam:Paris Bettencourt/Notebook/Drug Screening/Tuesday 6th August.html2013-10-05T01:32:27Z<p>Idonnya: </p>
<hr />
<div><div dir="ltr"><br />
<b>Confirmation of strains SD002 - SD018 using colony PCR </b><br />
</div><br />
<div dir="ltr"><br />
Fresh single colonies of strains SD002-SD018 were prepared for PCR using the colony pcr protocol. The primers used for were FD_F, FD_R, FNR_F, FNR_R and SirA_F and SirA_R for each strain.<br />
</div><br />
<div dir="ltr"><br />
Strains SD002-SD018 were confirmed. <br />
<img src="https://static.igem.org/mediawiki/2013/f/f3/SirA_PCR_confirmation_.png" width="300" height="300"><br />
</div><br />
<div dir="ltr"><br />
<img src="https://static.igem.org/mediawiki/2013/b/b9/FNR_PCR_confirmation.png" width="300" height="300"><br />
</div><br />
<div dir="ltr"><br />
<img src="https://static.igem.org/mediawiki/2013/8/82/Colony_PCR_FD.png" width="300" height="300"><br />
</div></div>Idonnyahttp://2013.igem.org/File:Colony_PCR_FD.pngFile:Colony PCR FD.png2013-10-05T01:31:45Z<p>Idonnya: </p>
<hr />
<div></div>Idonnyahttp://2013.igem.org/File:FNR_PCR_confirmation.pngFile:FNR PCR confirmation.png2013-10-05T01:23:49Z<p>Idonnya: Colony PCR using FNR_F/R primers.</p>
<hr />
<div>Colony PCR using FNR_F/R primers.</div>Idonnyahttp://2013.igem.org/File:SirA_PCR_confirmation_.pngFile:SirA PCR confirmation .png2013-10-05T01:18:50Z<p>Idonnya: PCR confirmation of SirA</p>
<hr />
<div>PCR confirmation of SirA</div>Idonnyahttp://2013.igem.org/Team:Paris_Bettencourt/Notebook/Drug_Screening/Friday_30th_August.htmlTeam:Paris Bettencourt/Notebook/Drug Screening/Friday 30th August.html2013-10-05T00:51:15Z<p>Idonnya: </p>
<hr />
<div><html><br />
<div><br />
<b>Confirmation of P1 phage transduction:</b><br />
</div><br />
<p><br />
Colony PCR was used on patched colonies from the p1 phage transduction. The primers used for each colony were CysI F/R and KanR F/R.<br />
</p><br />
<div><br />
PCR confirmed that CysI was deleted in BL21(AI) and replaced with a Kanomycin resistance gene.<br />
<img src="https://static.igem.org/mediawiki/2013/f/f4/PCR_confirmation_of_BL21AIdeleted_CysI.jpg" width="300" height="300"><br />
</div><br />
<div><br />
2 Single colonies from each agar plate of the patched p1 phage transduced plates were picked and used to inoculate 5ml LB broth overnight. <br />
750ml of overnight culture was added to 250ml of 60% glycerol in a cryotube.<br />
</html></div>Idonnyahttp://2013.igem.org/Team:Paris_Bettencourt/Notebook/Drug_Screening/Tuesday_6th_August.htmlTeam:Paris Bettencourt/Notebook/Drug Screening/Tuesday 6th August.html2013-10-05T00:49:04Z<p>Idonnya: Created page with "<div dir="ltr"> <b>Confirmation of strains SD002 - SD018 using colony PCR </b> </div> <div dir="ltr"> Fresh single colonies of strains SD002-SD018 were prepared for PCR using..."</p>
<hr />
<div><div dir="ltr"><br />
<b>Confirmation of strains SD002 - SD018 using colony PCR </b><br />
</div><br />
<div dir="ltr"><br />
Fresh single colonies of strains SD002-SD018 were prepared for PCR using the colony pcr protocol. The primers used for were FD_F, FD_R, FNR_F, FNR_R and SirA_F and SirA_R for each strain.<br />
</div><br />
<div dir="ltr"><br />
Strains SD002-SD018 were confirmed. <br />
</div></div>Idonnyahttp://2013.igem.org/Team:Paris_Bettencourt/Notebook/Drug_Screening/Friday_30th_August.htmlTeam:Paris Bettencourt/Notebook/Drug Screening/Friday 30th August.html2013-10-05T00:34:38Z<p>Idonnya: </p>
<hr />
<div><html><br />
<div><br />
<b>Confirmation of P1 phage transduction:</b><br />
</div><br />
<p><br />
Colony PCR was used on patched colonies from the p1 phage transduction.<br />
</p><br />
<div><br />
PCR confirmed that CysI was deleted in BL21(AI) and replaced with a Kanomycin resistance gene.<br />
<img src="https://static.igem.org/mediawiki/2013/f/f4/PCR_confirmation_of_BL21AIdeleted_CysI.jpg" width="300" height="300"><br />
</div><br />
<div><br />
2 Single colonies from each agar plate of the patched p1 phage transduced plates were picked and used to inoculate 5ml LB broth overnight. <br />
750ml of overnight culture was added to 250ml of 60% glycerol in a cryotube.<br />
</html></div>Idonnyahttp://2013.igem.org/Team:Paris_Bettencourt/Notebook/Drug_Screening/Monday_5th_August.htmlTeam:Paris Bettencourt/Notebook/Drug Screening/Monday 5th August.html2013-10-05T00:25:53Z<p>Idonnya: </p>
<hr />
<div><div dir="ltr"><br />
<b>Confirmation of strains SD002 - SD018</b><br />
</div><br />
<div dir="ltr"><br />
Strains sd002-sd018 were grown overnight in 5ml LB.<br />
</div></div>Idonnyahttp://2013.igem.org/Team:Paris_Bettencourt/Notebook/Drug_Screening/Friday_30th_August.htmlTeam:Paris Bettencourt/Notebook/Drug Screening/Friday 30th August.html2013-10-05T00:08:48Z<p>Idonnya: </p>
<hr />
<div><html><br />
<div><br />
<b>Confirmation of P1 phage transduction:</b><br />
</div><br />
<p><br />
Colony PCR was used on patched colonies from the p1 phage transduction.<br />
</p><br />
<div><br />
PCR confirmed that CysI was deleted in BL21(AI) and replaced with a Kanomycin resistance gene.<br />
<img src="https://static.igem.org/mediawiki/2013/f/f4/PCR_confirmation_of_BL21AIdeleted_CysI.jpg" width="300" height="300"><br />
</div><br />
</html></div>Idonnyahttp://2013.igem.org/Team:Paris_Bettencourt/Notebook/Drug_Screening/Friday_30th_August.htmlTeam:Paris Bettencourt/Notebook/Drug Screening/Friday 30th August.html2013-10-05T00:05:48Z<p>Idonnya: </p>
<hr />
<div><html><br />
<div><br />
<b>Confirmation of P1 phage transduction:</b><br />
</div><br />
<p><br />
Colony PCR was used on patched colonies from the p1 phage transduction.<br />
</p><br />
<div><br />
PCR confirmed that CysI was deleted in BL21(AI) and replaced with a Kanomycin resistance gene.<br />
<img src="https://static.igem.org/mediawiki/2013/d/d4/PCR_confirmation_of_BL21AIdeleted_CysI.jpg" width="400" height="400"><br />
</div><br />
</html></div>Idonnyahttp://2013.igem.org/Team:Paris_Bettencourt/Notebook/Drug_Screening/Friday_30th_August.htmlTeam:Paris Bettencourt/Notebook/Drug Screening/Friday 30th August.html2013-10-04T23:51:47Z<p>Idonnya: </p>
<hr />
<div><div><br />
<b>Confirmation of P1 phage transduction:</b><br />
</div><br />
<p><br />
Colony PCR was used on patched colonies from the p1 phage transduction.<br />
</p><br />
<div><br />
PCR confirmed that CysI was deleted in BL21(AI) and replaced with a Kanomycin resistance gene.<br />
</p><br />
<img src="https://static.igem.org/mediawiki/2013/d/d4/PCR_confirmation_of_BL21AIdeleted_CysI.jpg" width="200" height="200"><br />
</div></div>Idonnyahttp://2013.igem.org/Team:Paris_Bettencourt/Notebook/Drug_Screening/Friday_30th_August.htmlTeam:Paris Bettencourt/Notebook/Drug Screening/Friday 30th August.html2013-10-04T23:50:01Z<p>Idonnya: Created page with "<div> <b>Confirmation of P1 phage transduction:</b> </div> <p> Colony PCR was used on patched colonies from the p1 phage transduction. </p> <div> PCR confirmed that C..."</p>
<hr />
<div><div><br />
<b>Confirmation of P1 phage transduction:</b><br />
</div><br />
<p><br />
Colony PCR was used on patched colonies from the p1 phage transduction.<br />
</p><br />
<div><br />
PCR confirmed that CysI was deleted in BL21(AI) and replaced with a Kanomycin resistance gene.<br />
</p><br />
<img src="https://static.igem.org/mediawiki/2013/d/d4/PCR confirmation of BL21AIdeleted CysI.jpg" width="200" height="200"><br />
</div></div>Idonnyahttp://2013.igem.org/File:PCR_confirmation_of_BL21AIdeleted_CysI.jpgFile:PCR confirmation of BL21AIdeleted CysI.jpg2013-10-04T23:44:18Z<p>Idonnya: Colony PCR confirming the deletion of CysI in E.coli BL21(AI).</p>
<hr />
<div>Colony PCR confirming the deletion of CysI in E.coli BL21(AI).</div>Idonnyahttp://2013.igem.org/Team:Paris_Bettencourt/Notebook/Drug_Screening/Thursday_29th_August.htmlTeam:Paris Bettencourt/Notebook/Drug Screening/Thursday 29th August.html2013-10-04T23:42:40Z<p>Idonnya: Created page with "<p> Colonies from the P1 phage transduction grew on the Kanomycin plates. </p> <div> Selected colonies were patched onto Kan and LBA plates and left to grow overnight. </..."</p>
<hr />
<div><p><br />
Colonies from the P1 phage transduction grew on the Kanomycin plates.<br />
</p><br />
<div><br />
Selected colonies were patched onto Kan and LBA plates and left to grow overnight.<br />
</div></div>Idonnyahttp://2013.igem.org/Team:Paris_Bettencourt/Notebook/Drug_Screening/Wednesday_28th_August.htmlTeam:Paris Bettencourt/Notebook/Drug Screening/Wednesday 28th August.html2013-10-04T23:36:45Z<p>Idonnya: </p>
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<div><!-- === Modify from here === --><br />
<b>P1 phage transduction</b><br />
<!-- === To here === --><br />
<p><br />
The transduction section of the P1 phage transduction protocol was carried out.<br />
</p><br />
<div><br />
Cells were plated on Kanamyocin plates to select for resistance and grown overnight.<br />
</div></div>Idonnyahttp://2013.igem.org/Team:Paris_Bettencourt/Notebook/Drug_Screening/Wednesday_28th_August.htmlTeam:Paris Bettencourt/Notebook/Drug Screening/Wednesday 28th August.html2013-10-04T23:33:46Z<p>Idonnya: Created page with "<p> The transduction section of the P1 phage transduction protocol was carried out. </p> <div> Cells were plated on Kanamyocin plates to select for resistance and grown o..."</p>
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<div><p><br />
The transduction section of the P1 phage transduction protocol was carried out.<br />
</p><br />
<div><br />
Cells were plated on Kanamyocin plates to select for resistance and grown overnight.<br />
</div></div>Idonnyahttp://2013.igem.org/Team:Paris_Bettencourt/Notebook/Drug_Screening/Tuesday_27th_August.htmlTeam:Paris Bettencourt/Notebook/Drug Screening/Tuesday 27th August.html2013-10-04T23:27:56Z<p>Idonnya: </p>
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<div class ="tbnote"><br />
<h2>Target</h2><br />
<a href="https://2013.igem.org/Team:Paris_Bettencourt/Project/Target" target="_blank" class="tbnotelogo DSlogo"> ASDF </a><br />
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<h3>Day Number<sup>suffix</sup> Month</h3><br />
<p><b><em><br />
<!-- === Modify from here === --><br />
Sequencing FdxA<br />
<!-- === To here === --><br />
</br></em></b></p><br />
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pJET FdxA sent for sequecing<br />
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<div dir="ltr"><br />
Steps 2-7 of the P1 phage transduction: Lysate preparation were carried out.<br />
</div><br />
<div dir="ltr"><br />
BL21(AI) was picked and placed into 5ml LB and incubated at 37C overnight.<br />
</div></div>Idonnyahttp://2013.igem.org/Team:Paris_Bettencourt/Notebook/Drug_Screening/Monday_26th_August.htmlTeam:Paris Bettencourt/Notebook/Drug Screening/Monday 26th August.html2013-10-04T23:18:48Z<p>Idonnya: Created page with "<div dir="ltr"> <b>P1 phage transduction</b> </div> <div dir="ltr"> The p1 phage lysate was prepared as described in the P1 phage transduction protocol. </div> <div dir="..."</p>
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<div><div dir="ltr"><br />
<b>P1 phage transduction</b><br />
</div><br />
<div dir="ltr"><br />
The p1 phage lysate was prepared as described in the P1 phage transduction protocol.<br />
</div><br />
<div dir="ltr"><br />
BL21 (DE3) <i>ΔCysI Δfpr ΔydbK </i>was grown in 5ml Lb.<br />
<br/><br />
</div></div>Idonnyahttp://2013.igem.org/Team:Paris_Bettencourt/Notebook/Drug_Screening/Monday_5th_August.htmlTeam:Paris Bettencourt/Notebook/Drug Screening/Monday 5th August.html2013-10-04T22:54:26Z<p>Idonnya: Created page with "<div dir="ltr"> <b>PCR verification of strains SD002 - SD018</b> </div> <div dir="ltr"> Strains sd002-sd018 were verified using the colony pcr protocol and FD, FNR and Si..."</p>
<hr />
<div><div dir="ltr"><br />
<b>PCR verification of strains SD002 - SD018</b><br />
</div><br />
<div dir="ltr"><br />
Strains sd002-sd018 were verified using the colony pcr protocol and FD, FNR and SirA primers.<br />
</div></div>Idonnyahttp://2013.igem.org/Team:Paris_Bettencourt/ProtocolsTeam:Paris Bettencourt/Protocols2013-10-04T21:44:43Z<p>Idonnya: </p>
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<h2> <b> Protocol 1:</b> Heat Shock Transformation of <i>E. coli</i></h2><br />
<p>This protocol can be used to transform chemically competent (i.e. from CaCl2) with a miniprepped plasmid or a ligation product.</p><br />
<h5>Note: Never vortex competent cells. Mix cells by gentle shaking.</h5><br />
<ol><br />
<li>Thaw competent cells on ice. These can be prepared using the CaCl2 protocol.</li><br />
<li>Place 20 ul of cells in a pre-chilled Eppendorf tube.<br />
<ul><br />
<li><u>For an Intact Vector:</u> Add 0.5 ul or less to the chilled cells</li><br />
<li><u>For a Ligation Product:</u> Add 2-3 ul to the chilled cells.</li><br />
</ul><br />
</li><br />
<li> Mix gently by flicking the tube.</li><br />
<li> Chill on ice for 10 minutes. <em>This step is optional, but can improve yields when transforming a ligation product.</em></li><br />
<li>Heat shock at 42 &deg;C for 30 seconds.</li><br />
<li>Return to ice for 2 minutes.</li><br />
<li>Add 200 ul LB medium and recover the cells by shaking at 37 &deg;C.<br /><br />
Another rich medium can substitute for the recovery.<br /><br />
The recovery time varies with the antibiotic selection.<br /><br />
Ampicillin: 15-30 minutes<br /><br />
Kanamycin or Spectinomycin: 30-60 minutes<br /><br />
Chloramphenicol: 60-120 minutes <br />
</li><br />
<li>Plate out the cells on selective LB.<br /><br />
Use glass beads to spread the cells.<br /><br />
The volume of cells plated depends on what is being transformed.<br /><br />
<ul><br />
<li><u>For an Intact Vector:</u> High transformation efficiencies are expected. Plating out 10 ul of recovered cells should produce many colonies.</li><br />
<li><u>For a Ligation Product:</u> Lower transformation efficiencies are expected. Therefore you can plate the entire 200 ul volume of recovered cells.</li><br />
</ul><br />
Note: 200 ul is the maximum volume of liquid that an LB plate can absorb.<br />
</li><br />
<li>Incubate at 37 &deg;C. Transformants should appear within 12 hrs.</li><br />
</ol><br />
<br /><br />
<br /><br />
<br />
<h2> <b> Protocol 2:</b> CaCl2 Competent Cells </h2><br />
<br />
<p>This protocol makes 4 ml of competent cells, and can be easily scaled up to make more. The cells are typically stored in 110 ul aliquots, so this will make about 35 tubes. A typical transformation uses 20 ul of cells.<br />
<br />
<h5> Note: Never vortex competent cells. Resuspend by pipetting with large Pasteur pipettes.</h5><br />
<br />
<ol><br />
<p><b><u>The night before:</u></b></p><br />
<li>The night before, inoculate a 5 ml culture and grow overnight with selection.<br />
<p><b><u>The day of:</u></b></p><br />
<li> Dilute cells ~ 1:200 into selective media.<br>For this example add 250 ul to 50 ml of selective media.<br>Note: The protocol is easily scaled to increase the number of cells.<br />
<li> Grow the cells to an OD600 of 0.6 – 0.7.<br />
<br>Use a large flask, 500ml, for good aeration.<br />
<br>Use a baffled flask for fastest growth.<br />
<br>This takes about 3 hours depending on the cells.<br />
<br>Medium-heavy cloudiness by eye is fine.<br />
<li>Spin down the cells at 4 ºC, 4000 rpm, 15 minutes.<br />
Note: Keep the cells at 4 ºC from now on.<br />
<li>Resuspend cells in 15 ml, ice-cold 100 mM CaCl2. <br />
Leave on ice 4 hours to overnight.<br />
<li>Spin down the cells at 4 ºC, 4000 rpm, 15 minutes.<br />
<li>Resuspend cells in 4 ml, ice-cold 100 mM CaCl2 + 15% glycerol.<br />
<li>Aliquot into pre-chilled Eppendorf tubes. Use immediately or store at -80ºC.<br /><br />
Note: Frozen cells are only good once.Do not refreeze cells once thawed.<br />
</ol><br />
<br /><br />
<br /><br />
</body><br />
<br />
<h2> <b> Protocol 3:</b> Glycerol Stocks </h2><br />
<br />
<ol><br />
<br />
<li>Pick Single colonies from agar plates<br />
<li>Innoculate 5ml LB broth overnight.<br />
<li>Add 750ml of overnight culture to 250ml of 60% glycerol in a cryotube.<br />
<li>Make two sets of Glycerol stocks freeze one at -20ºC and the other at -80ºC.<br />
</ol><br />
<br /><br />
<br /><br />
<br />
<h2> <b> Protocol 4: </b> Electroporation </h2><br />
<br />
<ol><br />
<b>Preparation of Electrocompetent Cells</b><br /><br />
Note: Competent cells should never be vortexted, as this will cause them to lyse <br /><br />
and release salts into the media. Resuspend cells by pipeting up and down with a large <br /><br />
pasteur pipet. Once they are chilled, cells should be continuously cold.<br /><br />
<br /><br />
<li>The night before the transformation, start an overnight culture of cells.<br /><br />
5 ml LB Amp.<br /><br />
<br /><br />
<li>The day of the transformation, dilute the cells 100X.<br /><br />
100 ml LB Amp.<br /><br />
Grow at 30&deg;C for about 90 minutes.<br /><br />
<br /><br />
<li>Harvest the cells.<br /><br />
When the cells reach an OD600 of between 0.6 and 0.8.<br /><br />
Split the culture into 2x 50 ml falcon tubes, on ice.<br /><br />
Centrifuge at 4 &deg;C for 10 min at 4000 rpm.<br /><br />
<br /><br />
<li>Wash and combine the cells.<br /><br />
Remove the supernatant.<br /><br />
Resuspend the cells in 2x 25 ml of ice cold water.<br /><br />
Combine the volumes in a single 50 ml falcon tube.<br /><br />
<br /><br />
<li>Wash the cells 2 more times.<br /><br />
Centrifuge at 4 &deg;C for 10 min at 4000 rpm.<br /><br />
Resuspend in 50 ml of ice cold water.<br /><br />
Repeat.<br /><br />
<br /><br />
<li>Wash and concentrate the cells for electroporation.<br /><br />
Centrifuge at 4 &deg;C for 10 min at 4000 rpm.<br /><br />
Resuspend in 1-2 ml of ice cold water.<br /><br />
We will use 200 ul of washed cells per transformation.<br /><br />
</ol><br />
<ol><br />
<br /><br />
<b>Dialysis of PCR or Digestion Products</b><br /><br />
Note: DNA for electroporation must be free of salts to avoid arcing.<br /><br />
<br /><br />
<li>Float a filter in a Petri dish filled with water.<br /><br />
Millipore membrane filter 0.025 uM.<br /><br />
<br /><br />
<li>Pipet one drop of PCR product onto the filter.<br /><br />
200 ng is needed per transformation.<br /><br />
20 - 100 ul fits well on one filter.<br /><br />
<br /><br />
<li>Collect the drop after 30 - 45 minutes.<br /><br />
The volume will change, but the DNA is not lost.<br />
</ol><br />
<br /><br />
<br /><br />
<br />
<h2> <b> Protocol 5:</b> Miniprep </h2><br />
<br />
<h3><b>Miniprep using <i>Thermo Scientific GeneJET Plasmid Miniprep Kit</i></b></h3><br />
<br /><br />
All centrifugations should be carried out in a table-top microcentrifuge at sup12000 x g<br /><br />
<br /><br />
<ol><br />
<li>Resuspend the pelleted cells in 250 ul of the Resuspension Solution. Transfer the cell suspension to a microcentrifuge tube. The bacteria should be resuspended completely by vortexing or pipetting up and down un<br />
til no cell clumps remain.<br />
<br /><br />
<b><i>Note</i></b>.<br />
Ensure RNase A has been added to the Resuspension Solution.<br /><br />
<br /><br />
<li>Add 250 ul of the Lysis Solution and mix thoroughly by inverting the tube 4-6 times until the solution becomes viscous and slightly clear.<br />
<br /><br />
<b><i>Note</i></b>. Do not vortex to avoid shearing of chromosomal DNA. Do not incubate for more than 5 min to avoid denaturation of supercoiled plasmid DNA.<br />
<br /><br />
<br /><br />
<li>Add 350 ul of the Neutralization Solution and mix immediately and thoroughly by inverting the tube 4-6 times.<br />
<br /><br />
<b><i>Note</i></b>.<br />
It is important to mix thoroughly and gently after the addition of the Neutralization Solution to avoid localized precipitation of bacterial cell debris. The neutralized bacterial lysate should become cloudy.<br />
<br /><br />
<br /><br />
<li>Centrifuge for 5 min to pellet cell debris and chromosomal DNA. <br />
<br /><br />
<br /><br />
<li>Transfer the supernatant to the supplied GeneJET spin column by decanting or pipetting. Avoid disturbing or transferring the white precipitate. <br />
<br /><br />
<br /><br />
<li>Centrifuge for 1 min. Discard the flow-through and place the column back into the same collection tube.<br />
<br /><br />
<b><i>Note</i></b>.<br />
Do not add bleach to the flow-through.<br />
<br /><br />
<br /><br />
<li>Add 500 ul of the Wash Solution (diluted with ethanol) to the GeneJET spin column. Centrifuge for 30-60 seconds and discard the flow-through. Place the column back into the same collection tube. <br />
<br /><br />
<br /><br />
<li>Repeat the wash procedure (step 7) using 500 ul of the Wash Solution. <br />
<br /><br />
<br /><br />
<li>Discard the flow-through and centrifuge for an additional 1 min to remove residual Wash Solution. This step is essential to avoid residual ethanol in plasmid preps. <br />
<br /><br />
<br /><br />
<li>Transfer the GeneJETspin column into a fresh 1.5 ml microcentrifuge tube. Add 50 ul of the Elution Bufferto the center of GeneJET spin column membrane to elute the plasmid DNA. Take care not to contact the membrane with the pipette tip. Incubate for 2 min at room tempera ture and centrifuge for 2 min.<br />
<br /><br />
<b><i>Note</i></b>.<br />
An additional elution step (<i>optional</i>) with Elution Buffer or water will recover residual DNA from the membrane and increase the overall yield by 10-20%. For elution of plasmids or cosmids sup20 kb, prewarm Elution Buffer to 70&deg;C before applying to silica membrane.<br />
<br /><br />
<br /><br />
<li>Discard the column and store the purified plasmid DNA at -20&deg;C. <br />
</ol><br />
<br /><br />
<br /><br />
<br />
<h2> <b> Protocol 6:</b> PCR Purification </h2><br />
<br />
<h3><b>PCR purification using <i>Thermo Scientific GeneJET PCR Purification Kit</i></b></h3><br />
<br /><br />
All centrifugations should be carried out in a table-top microcentrifuge at sup12000 x g<br /><br />
<br /><br />
<ol><br />
<li>Add a 1:1 volume of Binding Buffer to completed PCR mixture (e.g. for every 100 uL of reaction mixture, add 100 uL of Binding Buffer). Mix thoroughly. Check the color of the solution. A yellow color indicates an optimal pH for DNA binding. If the color of the solution is orange or violet, add 10 uL of 3 M sodium acetate, pH 5.2 solution and mix. The color of the mix will become yellow.<br />
<br /><br />
<br /><br />
<li><i>Optional</i>: if the DNA fragment is inf 500 bp, add a 1:2 volume of 100% isopropanol(e.g., 100 uL of isopropanol should be added to 100 uL of PCR mixture combined with 100 uL of Binding Buffer). Mix thoroughly.<br /><br />
<b><i>Note</i></b>. If PCR mixture contains primer-dimers, purification without isopropanol is recommended. However, the yield of the target DNA fragment will be lower.<br />
<br /><br />
<br /><br />
<li>Transfer up to 800 uL of the solution from step 1(or optional step 2)to the GeneJET<br />
purification column. Centrifuge for 30-60 s. Discard the flow-through.<br /><br />
<b><i>Note</i></b>. If the total volume exceeds 800 uL, the solution can be added to the column in stages. After the addition of 800 uL of solution, centrifuge the column for 30-60 s and discard flow-through. Repeat until the entire solution has been added to the column membrane.<br />
<br /><br />
<br /><br />
<li>Add 700 uL of Wash Buffer to the GeneJET purification column. Centrifuge for 30-60 s. Discard the flow-through and place the purification column back into the collection tube.<br />
<br /><br />
<br /><br />
<li>Centrifuge the empty GeneJET purification column for an additional 1 min to completely remove any residual wash buffer.<br /><br />
<b><i>Note</i></b>.This step is essential as the presence of residual ethanol in the DNA sample may inhibit subsequent reactions<br />
<br /><br />
<br /><br />
<li>Transfer the GeneJET purification column to a clean 1.5 mL microcentrifuge tube (not included).Add 50 uL of Elution Buffer to the center of the GeneJET purification column membrane and centrifuge for 1 min.<br /><br />
<b><i>Note</i></b>. For low DNA amounts the elution volumes can be reduced to increase DNA concentration. An elution volume between 20-50 uL does not significantly reduce the DNA yield. However, elution volumes less than 10 uL are not recommended. If DNA fragment is inf 10 kb, prewarm Elution Buffer to 65&deg;C before applying to column. If the elution volume is 10 uL and DNA amount is inf 5 ug, incubate column for 1 min at room temperature before centrifugation.<br />
<br /><br />
<br /><br />
<li>Discard the GeneJET purification column and store the purified DNA at -20&deg;C. <br />
</ol><br />
<br /><br />
<br /><br />
<br />
<h2> <b> Protocol 7:</b> Gel Purification </h2><br />
<br />
<h3><b>Gel purification using <i>Thermo Scientific GeneJET Gel Extraction Kit</i></b></h3><br />
<br /><br />
All centrifugations should be carried out in a table-top microcentrifuge at sup12000 x g<br /><br />
<br /><br />
<ol><br />
<li>Excise gel slice containing the DNA fragment using a clean scalpel or razor blade. Cut as close to the DNA as possible to minimize the gel volume. Place the gel slice into a pre-weighed 1.5 ml tube and weigh. Record the weight of the gel slice.<br />
<br /><br />
<b><i>Note</i></b>.<br />
If the purified fragment will be used for cloning reactions, avoid damaging the DNA through UV light exposure. Minimize UV exposure to a few seconds or keep the gel slice on a glass or plastic plate during UV illumination.<br />
<br /><br />
<br /><br />
<li>Add 1:1 volume of Binding Buffer to the gel slice (volume: weight)(e.g., add 100 ul of Binding Buffer for every 100 mg of agarose gel).<br />
<br /><br />
<b><i>Note</i></b>.<br />
For gels with an agarose content greater than 2%, a dd 2:1 volumes of Binding Buffer to the gel slice.<br />
<br /><br />
<br /><br />
<li>Incubate the gel mixture at 50-60&deg;C for 10 min or until the gel slice is completely dissolved. Mix the tube by inversion every few minutes to facilitate the melting process. Ensure that the gel is completely dissolved. Vortex the gel mixture briefly before loading on the column. Check the color of the solution. A yellow color indicates an optimal pH for DNA binding. If the color of the solution is orange or violet, add 10 ul of 3 M sodium acetate, pH 5.2 solution and mix. The color of the mix will become yellow.<br />
<br /><br />
<br /><br />
<li><i>Optional</i>: use this step only when DNA fragment is inf 500 bp or sup10 kb long. If the DNA fragment is inf 500 bp, add a 1:2 volume of 100% isopropanol to the so lubilized gel solution (e.g. 100 ul of isopropanol should be added to 100 mg gel slice solubilized in 100 ul of Binding Buffer). Mix thoroughly. If the DNA fragment is sup10 kb , add a 1:2 volume of water to the solubilized gel solution (e.g. 100 ul of water should be added to 100 mg gel slice solubilized in 100 ul of Binding Buffer). Mix thoroughly. <br />
<br /><br />
<br /><br />
<li>Transfer up to 800 ul of the solubilized gel solution (from step 3 or 4) to the GeneJET purification column. Centrifuge for 1 min. Discard the flow-through and place the column back into the same collection tube.<br />
<br /><br />
<b><i>Note</i></b>. If the total volume exceeds 800 ul, the solution can be added to the column in stages. After each application, centrifuge the column for 30-60 s and discard the flow-through aftereach spin. Repeat until the entire volume has been applied to the column membrane. Do not exceed 1 g of total agarose gel per column.<br />
<br /><br />
<br /><br />
<li><i>Optional</i>: use this additional binding step only if the purified DNA will be used for sequencing. Add 100 ul of Binding Buffer to the GeneJET purification column. Centrifuge for 1 min. Discard the flow-through and place the column back into the same collection tube.<br />
<br /><br />
<br /><br />
<li>Centrifuge the empty GeneJET purification column for an additional 1 min to completely remove residual wash buffer.<br />
<br /><br />
<b><i>Note</i></b>. This step is essential to avoid residual ethanol in the purified DNA solution. The presence of ethanol in the DNA sample may inhibit downstream enzymatic reactions.<br />
<br /><br />
<br /><br />
<li>Transfer the GeneJET purification column into a clean 1.5 ml microcentrifuge tube (not included). Add 50 ul of Elution Buffer to the center of the purification column membrane. Centrifuge for 1 min.<br />
<br /><br />
<b><i>Note</i></b>. For low DNA amounts the elution volumes can be reduced to increase DNA concentration. An elution volume between 20-50 ul does not significantly reduce the DNA yield. However, elution volumes less than 10 ul are not recommended. If DNA fragment is sup10 kb, prewarm Elution Buffer to 65&deg;C before applying to column. If the elution volume is 10 ul and DNA amount is inf5 ug, incubate column for 1 min at room temperature before centrifugation.<br />
<br /><br />
<br /><br />
<li>Discard the GeneJET purification column and store the purified DNA at -20&deg;C.<br />
</ol><br />
<br /><br />
<br /><br />
<br />
<div><br />
<h2> <b> Protocol 8: P1 Transduction</b></h2><br />
</div><br />
<div><br />
<br/><br />
</div><br />
<div><br />
This protocol uses a phage to transfer a marker from a donor strain to a recipient strain. The phage head packages about 90 kb of DNA, so donor DNA near<br />
the marker is also transferred. Note that this can cause problems if you are working with several markers that are very close together.<br />
</div><br />
<div><br />
<br/><br />
</div><br />
<div><br />
<strong><u>Lysate preparation</u></strong><br />
</div><br />
<div><br />
<br/><br />
</div><br />
<div><br />
<strong>Note: P1 phage should be stored at 4 C. It can't be frozen.</strong><br />
</div><br />
<div><br />
<br/><br />
</div><br />
<div><br />
1) The night before, start a 5 mL culture of the donor strain in selective LB.<br />
</div><br />
<div><br />
<br/><br />
</div><br />
<div><br />
2) The day of, dilute the donor strain 1:100 into Phage Lysis medium.<br />
</div><br />
<div><br />
50 ul of cells in 5 mL LB<br />
<br/><br />
</div><br />
<div><br />
+ 50 uL of 20% Glucose<br />
<br/><br />
</div><br />
<div><br />
+ 50 uL of 1M MgSO4<br />
<br/><br />
</div><br />
<div><br />
+ 25 ul of 1M CaCl2<br />
<br/><br />
</div><br />
<div><br />
No antibiotics<br />
<br/><br />
</div><br />
<div><br />
<br/><br />
</div><br />
<div><br />
3) Incubate at 37 C for 1 hour.<br />
<br/><br />
</div><br />
<div><br />
<br/><br />
</div><br />
<div><br />
4) Add 50 ul of P1 phage lysate.<br />
<br/><br />
</div><br />
<div><br />
Monitor the culture for 1-3 hours.<br />
<br/><br />
</div><br />
<div><br />
The culture should become cloudy, then clear following lysis.<br />
<br/><br />
</div><br />
<div><br />
<br/><br />
</div><br />
<div><br />
5) Add 500 ul of chloroform to the lysate and vortex.<br />
<br/><br />
</div><br />
<div><br />
<br/><br />
</div><br />
<div><br />
6) Centrifuge at max speed for 1 minute to clear the cell debris.<br />
<br/><br />
</div><br />
<div><br />
Collect the supernatant.<br />
</div><br />
<div><br />
<br/><br />
</div><br />
<div><br />
7) Phage lysate can be stored indefinitely at 4 C. Freezing will destroy the phage.<br />
</div><br />
<div><br />
</div><br />
<div><br />
<strong><u>Transduction</u></strong><br />
</div><br />
<div><br />
<br/><br />
</div><br />
<div><br />
1) The night before, start a 5 ml culture of the recipient strain in selective LB.<br />
</div><br />
<div><br />
<br/><br />
</div><br />
<div><br />
2) The day of, harvest the cells by centrifugation.<br />
<br/><br />
</div><br />
<div><br />
6000 rpm for 2 min.<br />
<br/><br />
</div><br />
<div><br />
<br/><br />
</div><br />
<div><br />
3) Resuspend in original culture volume in 5 mL Phage Infection LB.<br />
<br/><br />
</div><br />
<div><br />
5 mL LB<br />
<br/><br />
</div><br />
<div><br />
+ 50 uL of 1M MgSO4<br />
<br/><br />
</div><br />
<div><br />
+ 25 ul of 1M CaCl2<br />
<br/><br />
</div><br />
<div><br />
<br/><br />
</div><br />
<div><br />
4) Transfer 100 uL of donor P1 lysate per transformation to a 1.5 mL tube.<br />
<br/><br />
</div><br />
<div><br />
Incubate at 37 C for 30 minutes.<br />
<br/><br />
</div><br />
<div><br />
This allows the residual chloroform to evaporate.<br />
<br/><br />
</div><br />
<div><br />
<br/><br />
</div><br />
<div><br />
5) Set up 4 reactions for each transduction<br />
<br/><br />
</div><br />
<div><br />
1) 100 uL Donor Lysate 2) 10 uL Donor Lysate 3) 100 uL Donor Lysate 4) 100 uL Plain Lb<br />
<br/><br />
</div><br />
<div><br />
100 uL Recipient Cells 190 uL Recipient Cells 100 uL Plain LB 100 uL Recipient Cells<br />
</div><br />
<div><br />
<br/><br />
</div><br />
<div><br />
6) Incubate at 37 C for 30 minutes.<br />
<br/><br />
</div><br />
<div><br />
<br/><br />
</div><br />
<div><br />
7) Stop the infection with 200 uL of 1 M Sodium Citrate (pH 5.5).<br />
<br/><br />
</div><br />
<div><br />
<br/><br />
</div><br />
<div><br />
8) Add 1 mL LB and recover the cells for 1-2 hours.<br />
<br/><br />
</div><br />
<div><br />
<br/><br />
</div><br />
<div><br />
9) Spin the cells down and resuspend for plating.<br />
<br/><br />
</div><br />
<div><br />
100 ul LB + 10 uL of 1 M Sodium Citrate (pH 5.5)<br />
<br/><br />
</div><br />
<div><br />
10) Plate on selective LB.<br />
</div><br />
</ol><br />
<br /><br />
<br /><br />
<br />
<div><br />
<h2><b>Protocol 9: Colony PCR</b></h2><br />
<div><br />
<b><br />
<br/><br />
</b><br />
<div><br />
1. Pick a single colony into 30ul of nuclease-free H20. (Fresh colonies grown that day work best, but they can also come from 4C).<br />
</div><br />
<div><br />
<br/><br />
</div><br />
<div><br />
2. Boil for 10 minutes at 100C.<br />
</div><br />
<div><br />
<br/><br />
</div><br />
<div><br />
3. Centrifuge (find G) for 1 min. 1ul of this can be used directly for PCR. Best if used directly, but can also be stored at 4C for a few days.<br />
<div><br />
<p dir="ltr"><br />
<b>PCR Reaction</b><br />
</p><br />
<p dir="ltr"><br />
Keep all the reagents at 4C while preparing the mixture. Pre-heat the thermocycler to 95C and transfer your reaction directly from 4C.<br />
</p><br />
<p dir="ltr"><br />
<br/><br />
</p><br />
<div dir="ltr"><br />
<table><br />
<colgroup><br />
<col width="163"/><br />
<col width="137"/><br />
<col width="153"/><br />
<col width="143"/><br />
</colgroup><br />
<tbody><br />
<tr><br />
<td><br />
<p dir="ltr"><br />
Reagent<br />
</p><br />
</td><br />
<td><br />
<p dir="ltr"><br />
Volume ul<br />
</p><br />
</td><br />
</tr><br />
<tr><br />
<td><br />
<p dir="ltr"><br />
Forward Primer<br />
</p><br />
</td><br />
<td><br />
<p dir="ltr"><br />
1.0<br />
</p><br />
</td><br />
</tr><br />
<tr><br />
<td><br />
<p dir="ltr"><br />
Reverse Primer<br />
</p><br />
</td><br />
<td><br />
<p dir="ltr"><br />
1.0<br />
</p><br />
</td><br />
</tr><br />
<tr><br />
<td><br />
<p dir="ltr"><br />
Template DNA <br />
</p><br />
</td><br />
<td><br />
<p dir="ltr"><br />
2<br />
</p><br />
</td><br />
</tr><br />
<tr><br />
<td><br />
<p dir="ltr"><br />
Quick-Load Taq<br />
<br/><br />
2x Master Mix<br />
</p><br />
</td><br />
<td><br />
<p dir="ltr"><br />
10<br />
</p><br />
</td><br />
</tr><br />
<tr><br />
<td><br />
<p dir="ltr"><br />
Nuclease-free water<br />
</p><br />
</td><br />
<td><br />
<p dir="ltr"><br />
6<br />
</p><br />
</td><br />
</tr><br />
<tr><br />
<td><br />
<p dir="ltr"><br />
Total Volume<br />
</p><br />
</td><br />
<td><br />
<p dir="ltr"><br />
20<br />
</p><br />
</td><br />
</tr><br />
</tbody><br />
</table><br />
</div><br />
<div><br />
<br/><br />
</div><br />
<div><br />
<br/><br />
</div><br />
<div><br />
<br/><br />
<div dir="ltr"><br />
<table><br />
<colgroup><br />
<col width="148"/><br />
<col width="124"/><br />
<col width="134"/><br />
<col width="114"/><br />
<col width="104"/><br />
</colgroup><br />
<tbody><br />
<tr><br />
<td><br />
<p dir="ltr"><br />
Thermocycler Protocol: NEB Quick-Load<br />
</p><br />
</td><br />
<td><br />
<br/><br />
</td><br />
<td><br />
<br/><br />
</td><br />
<td><br />
<br/><br />
</td><br />
<td><br />
<br/><br />
</td><br />
</tr><br />
<tr><br />
<td><br />
<br/><br />
</td><br />
<td><br />
<p dir="ltr"><br />
Temp:<br />
</p><br />
</td><br />
<td><br />
<p dir="ltr"><br />
Time<br />
</p><br />
</td><br />
<td><br />
<br/><br />
</td><br />
<td><br />
<br/><br />
</td><br />
</tr><br />
<tr><br />
<td><br />
<p dir="ltr"><br />
Start<br />
</p><br />
</td><br />
<td><br />
<p dir="ltr"><br />
95C<br />
</p><br />
</td><br />
<td><br />
<p dir="ltr"><br />
30sec<br />
</p><br />
</td><br />
<td><br />
<p dir="ltr"><br />
Melt<br />
</p><br />
</td><br />
<td><br />
<br/><br />
</td><br />
</tr><br />
<tr><br />
<td><br />
<br/><br />
</td><br />
<td><br />
<br/><br />
</td><br />
<td><br />
<br/><br />
</td><br />
<td><br />
<br/><br />
</td><br />
<td><br />
<br/><br />
</td><br />
</tr><br />
<tr><br />
<td><br />
<p dir="ltr"><br />
Cycle 1<br />
</p><br />
</td><br />
<td><br />
<p dir="ltr"><br />
95C<br />
</p><br />
</td><br />
<td><br />
<p dir="ltr"><br />
15sec<br />
</p><br />
</td><br />
<td><br />
<p dir="ltr"><br />
Melt<br />
</p><br />
</td><br />
<td><br />
<p dir="ltr"><br />
30 Cycles<br />
</p><br />
</td><br />
</tr><br />
<tr><br />
<td><br />
<p dir="ltr"><br />
Cycle 2<br />
</p><br />
</td><br />
<td><br />
<p dir="ltr"><br />
60C<br />
</p><br />
</td><br />
<td><br />
<p dir="ltr"><br />
30sec<br />
</p><br />
</td><br />
<td><br />
<p dir="ltr"><br />
Anneal<br />
</p><br />
</td><br />
<td><br />
<p dir="ltr"><br />
30 Cycles<br />
</p><br />
</td><br />
</tr><br />
<tr><br />
<td><br />
<p dir="ltr"><br />
Cycle 3<br />
</p><br />
</td><br />
<td><br />
<p dir="ltr"><br />
72C<br />
</p><br />
</td><br />
<td><br />
<p dir="ltr"><br />
1 min per kb<br />
</p><br />
</td><br />
<td><br />
<p dir="ltr"><br />
Extend<br />
</p><br />
</td><br />
<td><br />
<p dir="ltr"><br />
30 Cycles<br />
</p><br />
</td><br />
</tr><br />
<tr><br />
<td><br />
<br/><br />
</td><br />
<td><br />
<br/><br />
</td><br />
<td><br />
<br/><br />
</td><br />
<td><br />
<br/><br />
</td><br />
<td><br />
<br/><br />
</td><br />
</tr><br />
<tr><br />
<td><br />
<p dir="ltr"><br />
Finish<br />
</p><br />
</td><br />
<td><br />
<p dir="ltr"><br />
72C<br />
</p><br />
</td><br />
<td><br />
<p dir="ltr"><br />
5 min<br />
</p><br />
</td><br />
<td><br />
<p dir="ltr"><br />
Extend<br />
</p><br />
</td><br />
<td><br />
<br/><br />
</td><br />
</tr><br />
<tr><br />
<td><br />
<p dir="ltr"><br />
Store<br />
</p><br />
</td><br />
<td><br />
<p dir="ltr"><br />
10C<br />
</p><br />
</td><br />
<td><br />
<p dir="ltr"><br />
Forever<br />
</p><br />
</td><br />
<td><br />
<p dir="ltr"><br />
Store<br />
</p><br />
</td><br />
<td><br />
<br/><br />
</td><br />
</tr><br />
</tbody><br />
</table><br />
</div><br />
<div dir="ltr"><br />
<p dir="ltr"><br />
The annealing temperature may vary from 45-68C, depending mostly on your primer.<br />
</p><br />
Samples can then be used for gel electrophoresis.<br />
<br/><br />
</div><br />
</div><br />
</div><br />
</div><br />
</div><br />
</ol><br />
<br /><br />
<br /><br />
<br />
<h2><b>Protocol 10: Preparation of LB liquid broth:</b></h2><br />
</div><br />
<p dir="ltr"><br />
LB is our standard rich media. Directions are on the bottle.<br />
</p><br />
<p dir="ltr"><br />
1. Add 12.5g of LB broth powder to a 1L glass bottle.<br />
</p><br />
<p dir="ltr"><br />
2. Add 500ml of osmotic water.<br />
</p><br />
<p dir="ltr"><br />
3. Autoclave <br />
</p><br />
<p dir="ltr"><br />
<br/><br />
</p><br />
<div dir="ltr"><br />
<br/><br />
</div><br />
</ol><br />
<br /><br />
<br /><br />
<br />
<h2><b>Protocol 11: Preparation of LB Agar:</b></h2><br />
<div dir="ltr"><br />
</div><br />
<p dir="ltr"><br />
LB agar is our standard rich media for plating. Directions are again on the bottle.<br />
</p><br />
<p dir="ltr"><br />
1. Add 20 g of LB agar powder to a 1 L glass bottle.<br />
</p><br />
<p dir="ltr"><br />
2. Add 500 ml of osmotic water.<br />
</p><br />
<p dir="ltr"><br />
3. Autoclave<br />
<br/><br />
</p><br />
<p dir="ltr"><br />
<br/><br />
</p><br />
<div dir="ltr"><br />
<br/><br />
</div><br />
</ol><br />
<br /><br />
<br /><br />
<br />
<div dir="ltr"><br />
<h2><b>Protocol 12: Making LB Agar plates:</b></h2><br />
</div><br />
<p dir="ltr"><br />
1. Melt LBA in microwave (~8 min).<br />
</p><br />
<p dir="ltr"><br />
2. Don’t burn yourself. Most Antibiotics are thermo sensitive so allow agar to cool to approximately 56º C before adding antibiotics.<br />
</p><br />
<p dir="ltr"><br />
3. Add antibiotics if required<br />
</p><br />
<p dir="ltr"><br />
4. Add 15-25 ml of LBA into each plate. You can get more plates with less agar in each plate, but the plates will desiccate faster and cannot be stored as<br />
long.<br />
</p><br />
<p dir="ltr"><br />
5. Allow the plates to set.<br />
</p><br />
<p dir="ltr"><br />
6. Dry them 45' under the flow hood, with the lid open.<br />
<br/><br />
</p><br />
<div dir="ltr"><br />
<br/><br />
</div><br />
</ol><br />
<br /><br />
<br /><br />
<br />
<div dir="ltr"><br />
<h2><b>Protocol 13: Gel Electrophoresis:</b></h2><br />
</div><br />
<div dir="ltr"><br />
Note: Making a standard 1% agarose gel<br />
</div><br />
<div dir="ltr"><br />
<br/><br />
</div><br />
<div dir="ltr"><br />
<p dir="ltr"><br />
Agarose gels are commonly used in concentrations between 0.7 to 2% depending on the size of bands needed to be separated. Simply adjust the amount of<br />
starting agarose to %g/100mL TAE e.g. 2g/100mL will give 2%.<br />
</p><br />
<p dir="ltr"><br />
</p><br />
<p dir="ltr"><br />
1. Measure out 1g of agarose<br />
</p><br />
<br/><br />
<p dir="ltr"><br />
2. Pour agarose powder into a microwavable flask along with 100mL of 1xTAE<br />
</p><br />
<br/><br />
<p dir="ltr"><br />
3. Microwave for 1-3mins (until the agarose has dissolved completely and there is a nice rolling boil). Caution HOT! Be careful stirring, eruptive<br />
boiling can occur.<br />
</p><br />
<br/><br />
<p dir="ltr"><br />
4. Let agarose solution cool down for 5min.<br />
</p><br />
<br/><br />
<p dir="ltr"><br />
5. Pour the agarose into a gel tray with the suitable well comb in place (pour slowly to avoid bubbles which will disrupt the gel).<br />
<br/><br />
</p><br />
<p dir="ltr"><br />
6. Place newly poured gel at 4°C for 10-15 minutes or let sit at room temperature for 20-30 minutes, until the gel has completely solidified.<br />
</p><br />
<br/><br />
<p dir="ltr"><br />
7. Once solidified, remove the comb and place the gel into the electrophoresis unit (gel box).<br />
</p><br />
<br/><br />
<p dir="ltr"><br />
8. Fill the gel box with 1xTAE buffer.<br />
</p><br />
<br/><br />
<p dir="ltr"><br />
9. Carefully load GeneRuler 100bp Plus DNA weight ladder into the first lane of the gel.<br />
</p><br />
<br/><br />
<p dir="ltr"><br />
10. Carefully load your samples into the additional wells of the gel.<br />
</p><br />
<br/><br />
<p dir="ltr"><br />
11. Run the gel at 50-150V until the dye line is approximately 75-80% of the way down the gel.<br />
</p><br />
<br/><br />
<p dir="ltr"><br />
12. Turn off the power, disconnect the electrodes from the power source and then carefully remove the gel from the gel box.<br />
</p><br />
<br/><br />
<p dir="ltr"><br />
13. Place the gel into a container filled with 100ml of 1xTAE running buffer and 5μL of EtBr for 5 minutes.<br />
</p><br />
<br/><br />
14. Use any device that has UV light to visualize the DNA fragments.<br />
</ol><br />
<br /><br />
<br /><br />
<br />
<br />
</div><br />
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{{:Team:Paris_Bettencourt/footer}}</div>Idonnyahttp://2013.igem.org/Team:Paris_Bettencourt/ProtocolsTeam:Paris Bettencourt/Protocols2013-10-04T21:40:46Z<p>Idonnya: </p>
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<h2> <b> Protocol 1:</b> Heat Shock Transformation of <i>E. coli</i></h2><br />
<p>This protocol can be used to transform chemically competent (i.e. from CaCl2) with a miniprepped plasmid or a ligation product.</p><br />
<h5>Note: Never vortex competent cells. Mix cells by gentle shaking.</h5><br />
<ol><br />
<li>Thaw competent cells on ice. These can be prepared using the CaCl2 protocol.</li><br />
<li>Place 20 ul of cells in a pre-chilled Eppendorf tube.<br />
<ul><br />
<li><u>For an Intact Vector:</u> Add 0.5 ul or less to the chilled cells</li><br />
<li><u>For a Ligation Product:</u> Add 2-3 ul to the chilled cells.</li><br />
</ul><br />
</li><br />
<li> Mix gently by flicking the tube.</li><br />
<li> Chill on ice for 10 minutes. <em>This step is optional, but can improve yields when transforming a ligation product.</em></li><br />
<li>Heat shock at 42 &deg;C for 30 seconds.</li><br />
<li>Return to ice for 2 minutes.</li><br />
<li>Add 200 ul LB medium and recover the cells by shaking at 37 &deg;C.<br /><br />
Another rich medium can substitute for the recovery.<br /><br />
The recovery time varies with the antibiotic selection.<br /><br />
Ampicillin: 15-30 minutes<br /><br />
Kanamycin or Spectinomycin: 30-60 minutes<br /><br />
Chloramphenicol: 60-120 minutes <br />
</li><br />
<li>Plate out the cells on selective LB.<br /><br />
Use glass beads to spread the cells.<br /><br />
The volume of cells plated depends on what is being transformed.<br /><br />
<ul><br />
<li><u>For an Intact Vector:</u> High transformation efficiencies are expected. Plating out 10 ul of recovered cells should produce many colonies.</li><br />
<li><u>For a Ligation Product:</u> Lower transformation efficiencies are expected. Therefore you can plate the entire 200 ul volume of recovered cells.</li><br />
</ul><br />
Note: 200 ul is the maximum volume of liquid that an LB plate can absorb.<br />
</li><br />
<li>Incubate at 37 &deg;C. Transformants should appear within 12 hrs.</li><br />
</ol><br />
<br /><br />
<br /><br />
<br />
<h2> <b> Protocol 2:</b> CaCl2 Competent Cells </h2><br />
<br />
<p>This protocol makes 4 ml of competent cells, and can be easily scaled up to make more. The cells are typically stored in 110 ul aliquots, so this will make about 35 tubes. A typical transformation uses 20 ul of cells.<br />
<br />
<h5> Note: Never vortex competent cells. Resuspend by pipetting with large Pasteur pipettes.</h5><br />
<br />
<ol><br />
<p><b><u>The night before:</u></b></p><br />
<li>The night before, inoculate a 5 ml culture and grow overnight with selection.<br />
<p><b><u>The day of:</u></b></p><br />
<li> Dilute cells ~ 1:200 into selective media.<br>For this example add 250 ul to 50 ml of selective media.<br>Note: The protocol is easily scaled to increase the number of cells.<br />
<li> Grow the cells to an OD600 of 0.6 – 0.7.<br />
<br>Use a large flask, 500ml, for good aeration.<br />
<br>Use a baffled flask for fastest growth.<br />
<br>This takes about 3 hours depending on the cells.<br />
<br>Medium-heavy cloudiness by eye is fine.<br />
<li>Spin down the cells at 4 ºC, 4000 rpm, 15 minutes.<br />
Note: Keep the cells at 4 ºC from now on.<br />
<li>Resuspend cells in 15 ml, ice-cold 100 mM CaCl2. <br />
Leave on ice 4 hours to overnight.<br />
<li>Spin down the cells at 4 ºC, 4000 rpm, 15 minutes.<br />
<li>Resuspend cells in 4 ml, ice-cold 100 mM CaCl2 + 15% glycerol.<br />
<li>Aliquot into pre-chilled Eppendorf tubes. Use immediately or store at -80ºC.<br /><br />
Note: Frozen cells are only good once.Do not refreeze cells once thawed.<br />
</ol><br />
<br /><br />
<br /><br />
</body><br />
<br />
<h2> <b> Protocol 3:</b> Glycerol Stocks </h2><br />
<br />
<ol><br />
<br />
<li>Pick Single colonies from agar plates<br />
<li>Innoculate 5ml LB broth overnight.<br />
<li>Add 750ml of overnight culture to 250ml of 60% glycerol in a cryotube.<br />
<li>Make two sets of Glycerol stocks freeze one at -20ºC and the other at -80ºC.<br />
</ol><br />
<br /><br />
<br /><br />
<br />
<h2> <b> Protocol 4: </b> Electroporation </h2><br />
<br />
<ol><br />
<b>Preparation of Electrocompetent Cells</b><br /><br />
Note: Competent cells should never be vortexted, as this will cause them to lyse <br /><br />
and release salts into the media. Resuspend cells by pipeting up and down with a large <br /><br />
pasteur pipet. Once they are chilled, cells should be continuously cold.<br /><br />
<br /><br />
<li>The night before the transformation, start an overnight culture of cells.<br /><br />
5 ml LB Amp.<br /><br />
<br /><br />
<li>The day of the transformation, dilute the cells 100X.<br /><br />
100 ml LB Amp.<br /><br />
Grow at 30&deg;C for about 90 minutes.<br /><br />
<br /><br />
<li>Harvest the cells.<br /><br />
When the cells reach an OD600 of between 0.6 and 0.8.<br /><br />
Split the culture into 2x 50 ml falcon tubes, on ice.<br /><br />
Centrifuge at 4 &deg;C for 10 min at 4000 rpm.<br /><br />
<br /><br />
<li>Wash and combine the cells.<br /><br />
Remove the supernatant.<br /><br />
Resuspend the cells in 2x 25 ml of ice cold water.<br /><br />
Combine the volumes in a single 50 ml falcon tube.<br /><br />
<br /><br />
<li>Wash the cells 2 more times.<br /><br />
Centrifuge at 4 &deg;C for 10 min at 4000 rpm.<br /><br />
Resuspend in 50 ml of ice cold water.<br /><br />
Repeat.<br /><br />
<br /><br />
<li>Wash and concentrate the cells for electroporation.<br /><br />
Centrifuge at 4 &deg;C for 10 min at 4000 rpm.<br /><br />
Resuspend in 1-2 ml of ice cold water.<br /><br />
We will use 200 ul of washed cells per transformation.<br /><br />
</ol><br />
<ol><br />
<br /><br />
<b>Dialysis of PCR or Digestion Products</b><br /><br />
Note: DNA for electroporation must be free of salts to avoid arcing.<br /><br />
<br /><br />
<li>Float a filter in a Petri dish filled with water.<br /><br />
Millipore membrane filter 0.025 uM.<br /><br />
<br /><br />
<li>Pipet one drop of PCR product onto the filter.<br /><br />
200 ng is needed per transformation.<br /><br />
20 - 100 ul fits well on one filter.<br /><br />
<br /><br />
<li>Collect the drop after 30 - 45 minutes.<br /><br />
The volume will change, but the DNA is not lost.<br />
</ol><br />
<br /><br />
<br /><br />
<br />
<h2> <b> Protocol 5:</b> Miniprep </h2><br />
<br />
<h3><b>Miniprep using <i>Thermo Scientific GeneJET Plasmid Miniprep Kit</i></b></h3><br />
<br /><br />
All centrifugations should be carried out in a table-top microcentrifuge at sup12000 x g<br /><br />
<br /><br />
<ol><br />
<li>Resuspend the pelleted cells in 250 ul of the Resuspension Solution. Transfer the cell suspension to a microcentrifuge tube. The bacteria should be resuspended completely by vortexing or pipetting up and down un<br />
til no cell clumps remain.<br />
<br /><br />
<b><i>Note</i></b>.<br />
Ensure RNase A has been added to the Resuspension Solution.<br /><br />
<br /><br />
<li>Add 250 ul of the Lysis Solution and mix thoroughly by inverting the tube 4-6 times until the solution becomes viscous and slightly clear.<br />
<br /><br />
<b><i>Note</i></b>. Do not vortex to avoid shearing of chromosomal DNA. Do not incubate for more than 5 min to avoid denaturation of supercoiled plasmid DNA.<br />
<br /><br />
<br /><br />
<li>Add 350 ul of the Neutralization Solution and mix immediately and thoroughly by inverting the tube 4-6 times.<br />
<br /><br />
<b><i>Note</i></b>.<br />
It is important to mix thoroughly and gently after the addition of the Neutralization Solution to avoid localized precipitation of bacterial cell debris. The neutralized bacterial lysate should become cloudy.<br />
<br /><br />
<br /><br />
<li>Centrifuge for 5 min to pellet cell debris and chromosomal DNA. <br />
<br /><br />
<br /><br />
<li>Transfer the supernatant to the supplied GeneJET spin column by decanting or pipetting. Avoid disturbing or transferring the white precipitate. <br />
<br /><br />
<br /><br />
<li>Centrifuge for 1 min. Discard the flow-through and place the column back into the same collection tube.<br />
<br /><br />
<b><i>Note</i></b>.<br />
Do not add bleach to the flow-through.<br />
<br /><br />
<br /><br />
<li>Add 500 ul of the Wash Solution (diluted with ethanol) to the GeneJET spin column. Centrifuge for 30-60 seconds and discard the flow-through. Place the column back into the same collection tube. <br />
<br /><br />
<br /><br />
<li>Repeat the wash procedure (step 7) using 500 ul of the Wash Solution. <br />
<br /><br />
<br /><br />
<li>Discard the flow-through and centrifuge for an additional 1 min to remove residual Wash Solution. This step is essential to avoid residual ethanol in plasmid preps. <br />
<br /><br />
<br /><br />
<li>Transfer the GeneJETspin column into a fresh 1.5 ml microcentrifuge tube. Add 50 ul of the Elution Bufferto the center of GeneJET spin column membrane to elute the plasmid DNA. Take care not to contact the membrane with the pipette tip. Incubate for 2 min at room tempera ture and centrifuge for 2 min.<br />
<br /><br />
<b><i>Note</i></b>.<br />
An additional elution step (<i>optional</i>) with Elution Buffer or water will recover residual DNA from the membrane and increase the overall yield by 10-20%. For elution of plasmids or cosmids sup20 kb, prewarm Elution Buffer to 70&deg;C before applying to silica membrane.<br />
<br /><br />
<br /><br />
<li>Discard the column and store the purified plasmid DNA at -20&deg;C. <br />
</ol><br />
<br /><br />
<br /><br />
<br />
<h2> <b> Protocol 6:</b> PCR Purification </h2><br />
<br />
<h3><b>PCR purification using <i>Thermo Scientific GeneJET PCR Purification Kit</i></b></h3><br />
<br /><br />
All centrifugations should be carried out in a table-top microcentrifuge at sup12000 x g<br /><br />
<br /><br />
<ol><br />
<li>Add a 1:1 volume of Binding Buffer to completed PCR mixture (e.g. for every 100 uL of reaction mixture, add 100 uL of Binding Buffer). Mix thoroughly. Check the color of the solution. A yellow color indicates an optimal pH for DNA binding. If the color of the solution is orange or violet, add 10 uL of 3 M sodium acetate, pH 5.2 solution and mix. The color of the mix will become yellow.<br />
<br /><br />
<br /><br />
<li><i>Optional</i>: if the DNA fragment is inf 500 bp, add a 1:2 volume of 100% isopropanol(e.g., 100 uL of isopropanol should be added to 100 uL of PCR mixture combined with 100 uL of Binding Buffer). Mix thoroughly.<br /><br />
<b><i>Note</i></b>. If PCR mixture contains primer-dimers, purification without isopropanol is recommended. However, the yield of the target DNA fragment will be lower.<br />
<br /><br />
<br /><br />
<li>Transfer up to 800 uL of the solution from step 1(or optional step 2)to the GeneJET<br />
purification column. Centrifuge for 30-60 s. Discard the flow-through.<br /><br />
<b><i>Note</i></b>. If the total volume exceeds 800 uL, the solution can be added to the column in stages. After the addition of 800 uL of solution, centrifuge the column for 30-60 s and discard flow-through. Repeat until the entire solution has been added to the column membrane.<br />
<br /><br />
<br /><br />
<li>Add 700 uL of Wash Buffer to the GeneJET purification column. Centrifuge for 30-60 s. Discard the flow-through and place the purification column back into the collection tube.<br />
<br /><br />
<br /><br />
<li>Centrifuge the empty GeneJET purification column for an additional 1 min to completely remove any residual wash buffer.<br /><br />
<b><i>Note</i></b>.This step is essential as the presence of residual ethanol in the DNA sample may inhibit subsequent reactions<br />
<br /><br />
<br /><br />
<li>Transfer the GeneJET purification column to a clean 1.5 mL microcentrifuge tube (not included).Add 50 uL of Elution Buffer to the center of the GeneJET purification column membrane and centrifuge for 1 min.<br /><br />
<b><i>Note</i></b>. For low DNA amounts the elution volumes can be reduced to increase DNA concentration. An elution volume between 20-50 uL does not significantly reduce the DNA yield. However, elution volumes less than 10 uL are not recommended. If DNA fragment is inf 10 kb, prewarm Elution Buffer to 65&deg;C before applying to column. If the elution volume is 10 uL and DNA amount is inf 5 ug, incubate column for 1 min at room temperature before centrifugation.<br />
<br /><br />
<br /><br />
<li>Discard the GeneJET purification column and store the purified DNA at -20&deg;C. <br />
</ol><br />
<br /><br />
<br /><br />
<br />
<h2> <b> Protocol 7:</b> Gel Purification </h2><br />
<br />
<h3><b>Gel purification using <i>Thermo Scientific GeneJET Gel Extraction Kit</i></b></h3><br />
<br /><br />
All centrifugations should be carried out in a table-top microcentrifuge at sup12000 x g<br /><br />
<br /><br />
<ol><br />
<li>Excise gel slice containing the DNA fragment using a clean scalpel or razor blade. Cut as close to the DNA as possible to minimize the gel volume. Place the gel slice into a pre-weighed 1.5 ml tube and weigh. Record the weight of the gel slice.<br />
<br /><br />
<b><i>Note</i></b>.<br />
If the purified fragment will be used for cloning reactions, avoid damaging the DNA through UV light exposure. Minimize UV exposure to a few seconds or keep the gel slice on a glass or plastic plate during UV illumination.<br />
<br /><br />
<br /><br />
<li>Add 1:1 volume of Binding Buffer to the gel slice (volume: weight)(e.g., add 100 ul of Binding Buffer for every 100 mg of agarose gel).<br />
<br /><br />
<b><i>Note</i></b>.<br />
For gels with an agarose content greater than 2%, a dd 2:1 volumes of Binding Buffer to the gel slice.<br />
<br /><br />
<br /><br />
<li>Incubate the gel mixture at 50-60&deg;C for 10 min or until the gel slice is completely dissolved. Mix the tube by inversion every few minutes to facilitate the melting process. Ensure that the gel is completely dissolved. Vortex the gel mixture briefly before loading on the column. Check the color of the solution. A yellow color indicates an optimal pH for DNA binding. If the color of the solution is orange or violet, add 10 ul of 3 M sodium acetate, pH 5.2 solution and mix. The color of the mix will become yellow.<br />
<br /><br />
<br /><br />
<li><i>Optional</i>: use this step only when DNA fragment is inf 500 bp or sup10 kb long. If the DNA fragment is inf 500 bp, add a 1:2 volume of 100% isopropanol to the so lubilized gel solution (e.g. 100 ul of isopropanol should be added to 100 mg gel slice solubilized in 100 ul of Binding Buffer). Mix thoroughly. If the DNA fragment is sup10 kb , add a 1:2 volume of water to the solubilized gel solution (e.g. 100 ul of water should be added to 100 mg gel slice solubilized in 100 ul of Binding Buffer). Mix thoroughly. <br />
<br /><br />
<br /><br />
<li>Transfer up to 800 ul of the solubilized gel solution (from step 3 or 4) to the GeneJET purification column. Centrifuge for 1 min. Discard the flow-through and place the column back into the same collection tube.<br />
<br /><br />
<b><i>Note</i></b>. If the total volume exceeds 800 ul, the solution can be added to the column in stages. After each application, centrifuge the column for 30-60 s and discard the flow-through aftereach spin. Repeat until the entire volume has been applied to the column membrane. Do not exceed 1 g of total agarose gel per column.<br />
<br /><br />
<br /><br />
<li><i>Optional</i>: use this additional binding step only if the purified DNA will be used for sequencing. Add 100 ul of Binding Buffer to the GeneJET purification column. Centrifuge for 1 min. Discard the flow-through and place the column back into the same collection tube.<br />
<br /><br />
<br /><br />
<li>Centrifuge the empty GeneJET purification column for an additional 1 min to completely remove residual wash buffer.<br />
<br /><br />
<b><i>Note</i></b>. This step is essential to avoid residual ethanol in the purified DNA solution. The presence of ethanol in the DNA sample may inhibit downstream enzymatic reactions.<br />
<br /><br />
<br /><br />
<li>Transfer the GeneJET purification column into a clean 1.5 ml microcentrifuge tube (not included). Add 50 ul of Elution Buffer to the center of the purification column membrane. Centrifuge for 1 min.<br />
<br /><br />
<b><i>Note</i></b>. For low DNA amounts the elution volumes can be reduced to increase DNA concentration. An elution volume between 20-50 ul does not significantly reduce the DNA yield. However, elution volumes less than 10 ul are not recommended. If DNA fragment is sup10 kb, prewarm Elution Buffer to 65&deg;C before applying to column. If the elution volume is 10 ul and DNA amount is inf5 ug, incubate column for 1 min at room temperature before centrifugation.<br />
<br /><br />
<br /><br />
<li>Discard the GeneJET purification column and store the purified DNA at -20&deg;C.<br />
</ol><br />
<br /><br />
<br /><br />
<br />
<div><br />
<h2> <b> Protocol 8: P1 Transduction</b></h2><br />
</div><br />
<div><br />
<br/><br />
</div><br />
<div><br />
This protocol uses a phage to transfer a marker from a donor strain to a recipient strain. The phage head packages about 90 kb of DNA, so donor DNA near<br />
the marker is also transferred. Note that this can cause problems if you are working with several markers that are very close together.<br />
</div><br />
<div><br />
<br/><br />
</div><br />
<div><br />
<strong><u>Lysate preparation</u></strong><br />
</div><br />
<div><br />
<br/><br />
</div><br />
<div><br />
<strong>Note: P1 phage should be stored at 4 C. It can't be frozen.</strong><br />
</div><br />
<div><br />
<br/><br />
</div><br />
<div><br />
1) The night before, start a 5 mL culture of the donor strain in selective LB.<br />
</div><br />
<div><br />
<br/><br />
</div><br />
<div><br />
2) The day of, dilute the donor strain 1:100 into Phage Lysis medium.<br />
</div><br />
<div><br />
50 ul of cells in 5 mL LB<br />
<br/><br />
</div><br />
<div><br />
+ 50 uL of 20% Glucose<br />
<br/><br />
</div><br />
<div><br />
+ 50 uL of 1M MgSO4<br />
<br/><br />
</div><br />
<div><br />
+ 25 ul of 1M CaCl2<br />
<br/><br />
</div><br />
<div><br />
No antibiotics<br />
<br/><br />
</div><br />
<div><br />
<br/><br />
</div><br />
<div><br />
3) Incubate at 37 C for 1 hour.<br />
<br/><br />
</div><br />
<div><br />
<br/><br />
</div><br />
<div><br />
4) Add 50 ul of P1 phage lysate.<br />
<br/><br />
</div><br />
<div><br />
Monitor the culture for 1-3 hours.<br />
<br/><br />
</div><br />
<div><br />
The culture should become cloudy, then clear following lysis.<br />
<br/><br />
</div><br />
<div><br />
<br/><br />
</div><br />
<div><br />
5) Add 500 ul of chloroform to the lysate and vortex.<br />
<br/><br />
</div><br />
<div><br />
<br/><br />
</div><br />
<div><br />
6) Centrifuge at max speed for 1 minute to clear the cell debris.<br />
<br/><br />
</div><br />
<div><br />
Collect the supernatant.<br />
</div><br />
<div><br />
<br/><br />
</div><br />
<div><br />
7) Phage lysate can be stored indefinitely at 4 C. Freezing will destroy the phage.<br />
</div><br />
<div><br />
</div><br />
<div><br />
<strong><u>Transduction</u></strong><br />
</div><br />
<div><br />
<br/><br />
</div><br />
<div><br />
1) The night before, start a 5 ml culture of the recipient strain in selective LB.<br />
</div><br />
<div><br />
<br/><br />
</div><br />
<div><br />
2) The day of, harvest the cells by centrifugation.<br />
<br/><br />
</div><br />
<div><br />
6000 rpm for 2 min.<br />
<br/><br />
</div><br />
<div><br />
<br/><br />
</div><br />
<div><br />
3) Resuspend in original culture volume in 5 mL Phage Infection LB.<br />
<br/><br />
</div><br />
<div><br />
5 mL LB<br />
<br/><br />
</div><br />
<div><br />
+ 50 uL of 1M MgSO4<br />
<br/><br />
</div><br />
<div><br />
+ 25 ul of 1M CaCl2<br />
<br/><br />
</div><br />
<div><br />
<br/><br />
</div><br />
<div><br />
4) Transfer 100 uL of donor P1 lysate per transformation to a 1.5 mL tube.<br />
<br/><br />
</div><br />
<div><br />
Incubate at 37 C for 30 minutes.<br />
<br/><br />
</div><br />
<div><br />
This allows the residual chloroform to evaporate.<br />
<br/><br />
</div><br />
<div><br />
<br/><br />
</div><br />
<div><br />
5) Set up 4 reactions for each transduction<br />
<br/><br />
</div><br />
<div><br />
1) 100 uL Donor Lysate 2) 10 uL Donor Lysate 3) 100 uL Donor Lysate 4) 100 uL Plain Lb<br />
<br/><br />
</div><br />
<div><br />
100 uL Recipient Cells 190 uL Recipient Cells 100 uL Plain LB 100 uL Recipient Cells<br />
</div><br />
<div><br />
<br/><br />
</div><br />
<div><br />
6) Incubate at 37 C for 30 minutes.<br />
<br/><br />
</div><br />
<div><br />
<br/><br />
</div><br />
<div><br />
7) Stop the infection with 200 uL of 1 M Sodium Citrate (pH 5.5).<br />
<br/><br />
</div><br />
<div><br />
<br/><br />
</div><br />
<div><br />
8) Add 1 mL LB and recover the cells for 1-2 hours.<br />
<br/><br />
</div><br />
<div><br />
<br/><br />
</div><br />
<div><br />
9) Spin the cells down and resuspend for plating.<br />
<br/><br />
</div><br />
<div><br />
100 ul LB + 10 uL of 1 M Sodium Citrate (pH 5.5)<br />
<br/><br />
</div><br />
<div><br />
10) Plate on selective LB.<br />
</div><br />
</ol><br />
<br /><br />
<br /><br />
<br />
<div><br />
<h2><b>Protocol 9: Colony PCR</b></h2><br />
<div><br />
<b><br />
<br/><br />
</b><br />
<div><br />
1. Pick a single colony into 30ul of nuclease-free H20. (Fresh colonies grown that day work best, but they can also come from 4C).<br />
</div><br />
<div><br />
<br/><br />
</div><br />
<div><br />
2. Boil for 10 minutes at 100C.<br />
</div><br />
<div><br />
<br/><br />
</div><br />
<div><br />
3. Centrifuge (find G) for 1 min. 1ul of this can be used directly for PCR. Best if used directly, but can also be stored at 4C for a few days.<br />
<div><br />
<p dir="ltr"><br />
<b>PCR Reaction</b><br />
</p><br />
<p dir="ltr"><br />
Keep all the reagents at 4C while preparing the mixture. Pre-heat the thermocycler to 95C and transfer your reaction directly from 4C.<br />
</p><br />
<p dir="ltr"><br />
<br/><br />
</p><br />
<div dir="ltr"><br />
<table><br />
<colgroup><br />
<col width="163"/><br />
<col width="137"/><br />
<col width="153"/><br />
<col width="143"/><br />
</colgroup><br />
<tbody><br />
<tr><br />
<td><br />
<p dir="ltr"><br />
Reagent<br />
</p><br />
</td><br />
<td><br />
<p dir="ltr"><br />
Volume ul<br />
</p><br />
</td><br />
</tr><br />
<tr><br />
<td><br />
<p dir="ltr"><br />
Forward Primer<br />
</p><br />
</td><br />
<td><br />
<p dir="ltr"><br />
1.0<br />
</p><br />
</td><br />
</tr><br />
<tr><br />
<td><br />
<p dir="ltr"><br />
Reverse Primer<br />
</p><br />
</td><br />
<td><br />
<p dir="ltr"><br />
1.0<br />
</p><br />
</td><br />
</tr><br />
<tr><br />
<td><br />
<p dir="ltr"><br />
Template DNA <br />
</p><br />
</td><br />
<td><br />
<p dir="ltr"><br />
2<br />
</p><br />
</td><br />
</tr><br />
<tr><br />
<td><br />
<p dir="ltr"><br />
Quick-Load Taq<br />
<br/><br />
2x Master Mix<br />
</p><br />
</td><br />
<td><br />
<p dir="ltr"><br />
10<br />
</p><br />
</td><br />
</tr><br />
<tr><br />
<td><br />
<p dir="ltr"><br />
Nuclease-free water<br />
</p><br />
</td><br />
<td><br />
<p dir="ltr"><br />
6<br />
</p><br />
</td><br />
</tr><br />
<tr><br />
<td><br />
<p dir="ltr"><br />
Total Volume<br />
</p><br />
</td><br />
<td><br />
<p dir="ltr"><br />
20<br />
</p><br />
</td><br />
</tr><br />
</tbody><br />
</table><br />
</div><br />
<div><br />
<br/><br />
</div><br />
<div><br />
<br/><br />
</div><br />
<div><br />
<br/><br />
<div dir="ltr"><br />
<table><br />
<colgroup><br />
<col width="148"/><br />
<col width="124"/><br />
<col width="134"/><br />
<col width="114"/><br />
<col width="104"/><br />
</colgroup><br />
<tbody><br />
<tr><br />
<td><br />
<p dir="ltr"><br />
Thermocycler Protocol: NEB Quick-Load<br />
</p><br />
</td><br />
<td><br />
<br/><br />
</td><br />
<td><br />
<br/><br />
</td><br />
<td><br />
<br/><br />
</td><br />
<td><br />
<br/><br />
</td><br />
</tr><br />
<tr><br />
<td><br />
<br/><br />
</td><br />
<td><br />
<p dir="ltr"><br />
Temp:<br />
</p><br />
</td><br />
<td><br />
<p dir="ltr"><br />
Time<br />
</p><br />
</td><br />
<td><br />
<br/><br />
</td><br />
<td><br />
<br/><br />
</td><br />
</tr><br />
<tr><br />
<td><br />
<p dir="ltr"><br />
Start<br />
</p><br />
</td><br />
<td><br />
<p dir="ltr"><br />
95C<br />
</p><br />
</td><br />
<td><br />
<p dir="ltr"><br />
30sec<br />
</p><br />
</td><br />
<td><br />
<p dir="ltr"><br />
Melt<br />
</p><br />
</td><br />
<td><br />
<br/><br />
</td><br />
</tr><br />
<tr><br />
<td><br />
<br/><br />
</td><br />
<td><br />
<br/><br />
</td><br />
<td><br />
<br/><br />
</td><br />
<td><br />
<br/><br />
</td><br />
<td><br />
<br/><br />
</td><br />
</tr><br />
<tr><br />
<td><br />
<p dir="ltr"><br />
Cycle 1<br />
</p><br />
</td><br />
<td><br />
<p dir="ltr"><br />
95C<br />
</p><br />
</td><br />
<td><br />
<p dir="ltr"><br />
15sec<br />
</p><br />
</td><br />
<td><br />
<p dir="ltr"><br />
Melt<br />
</p><br />
</td><br />
<td><br />
<p dir="ltr"><br />
30 Cycles<br />
</p><br />
</td><br />
</tr><br />
<tr><br />
<td><br />
<p dir="ltr"><br />
Cycle 2<br />
</p><br />
</td><br />
<td><br />
<p dir="ltr"><br />
60C<br />
</p><br />
</td><br />
<td><br />
<p dir="ltr"><br />
30sec<br />
</p><br />
</td><br />
<td><br />
<p dir="ltr"><br />
Anneal<br />
</p><br />
</td><br />
<td><br />
<p dir="ltr"><br />
30 Cycles<br />
</p><br />
</td><br />
</tr><br />
<tr><br />
<td><br />
<p dir="ltr"><br />
Cycle 3<br />
</p><br />
</td><br />
<td><br />
<p dir="ltr"><br />
72C<br />
</p><br />
</td><br />
<td><br />
<p dir="ltr"><br />
1 min per kb<br />
</p><br />
</td><br />
<td><br />
<p dir="ltr"><br />
Extend<br />
</p><br />
</td><br />
<td><br />
<p dir="ltr"><br />
30 Cycles<br />
</p><br />
</td><br />
</tr><br />
<tr><br />
<td><br />
<br/><br />
</td><br />
<td><br />
<br/><br />
</td><br />
<td><br />
<br/><br />
</td><br />
<td><br />
<br/><br />
</td><br />
<td><br />
<br/><br />
</td><br />
</tr><br />
<tr><br />
<td><br />
<p dir="ltr"><br />
Finish<br />
</p><br />
</td><br />
<td><br />
<p dir="ltr"><br />
72C<br />
</p><br />
</td><br />
<td><br />
<p dir="ltr"><br />
5 min<br />
</p><br />
</td><br />
<td><br />
<p dir="ltr"><br />
Extend<br />
</p><br />
</td><br />
<td><br />
<br/><br />
</td><br />
</tr><br />
<tr><br />
<td><br />
<p dir="ltr"><br />
Store<br />
</p><br />
</td><br />
<td><br />
<p dir="ltr"><br />
10C<br />
</p><br />
</td><br />
<td><br />
<p dir="ltr"><br />
Forever<br />
</p><br />
</td><br />
<td><br />
<p dir="ltr"><br />
Store<br />
</p><br />
</td><br />
<td><br />
<br/><br />
</td><br />
</tr><br />
</tbody><br />
</table><br />
</div><br />
<div dir="ltr"><br />
<p dir="ltr"><br />
The annealing temperature may vary from 45-68C, depending mostly on your primer.<br />
</p><br />
Samples can then be used for gel electrophoresis.<br />
<br/><br />
</div><br />
</div><br />
</div><br />
</div><br />
</div><br />
</ol><br />
<br /><br />
<br /><br />
<br />
<h2><b>Protocol 10: Preparation of LB liquid broth:</b></h2><br />
</div><br />
<p dir="ltr"><br />
LB is our standard rich media. Directions are on the bottle.<br />
</p><br />
<p dir="ltr"><br />
1. Add 12.5g of LB broth powder to a 1L glass bottle.<br />
</p><br />
<p dir="ltr"><br />
2. Add 500ml of osmotic water.<br />
</p><br />
<p dir="ltr"><br />
3. Autoclave <br />
</p><br />
<p dir="ltr"><br />
<br/><br />
</p><br />
<div dir="ltr"><br />
<br/><br />
</div><br />
</ol><br />
<br /><br />
<br /><br />
<br />
<h2><b>Protocol 11: Preparation of LB Agar:</b></h2><br />
<div dir="ltr"><br />
</div><br />
<p dir="ltr"><br />
LB agar is our standard rich media for plating. Directions are again on the bottle.<br />
</p><br />
<p dir="ltr"><br />
1. Add 20 g of LB agar powder to a 1 L glass bottle.<br />
</p><br />
<p dir="ltr"><br />
2. Add 500 ml of osmotic water.<br />
</p><br />
<p dir="ltr"><br />
3. Autoclave<br />
<br/><br />
</p><br />
<p dir="ltr"><br />
<br/><br />
</p><br />
<div dir="ltr"><br />
<br/><br />
</div><br />
</ol><br />
<br /><br />
<br /><br />
<br />
<div dir="ltr"><br />
<h2><b>Protocol 12: Making LB Agar plates:</b></h2><br />
</div><br />
<p dir="ltr"><br />
1. Melt LBA in microwave (~8 min).<br />
</p><br />
<p dir="ltr"><br />
2. Don’t burn yourself. Most Antibiotics are thermo sensitive so allow agar to cool to approximately 56º C before adding antibiotics.<br />
</p><br />
<p dir="ltr"><br />
3. Add antibiotics if required<br />
</p><br />
<p dir="ltr"><br />
4. Add 15-25 ml of LBA into each plate. You can get more plates with less agar in each plate, but the plates will desiccate faster and cannot be stored as<br />
long.<br />
</p><br />
<p dir="ltr"><br />
5. Allow the plates to set.<br />
</p><br />
<p dir="ltr"><br />
6. Dry them 45' under the flow hood, with the lid open.<br />
<br/><br />
</p><br />
<div dir="ltr"><br />
<br/><br />
</div><br />
</ol><br />
<br /><br />
<br /><br />
<br />
</div><br />
</html><br />
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{{:Team:Paris_Bettencourt/footer}}</div>Idonnyahttp://2013.igem.org/Team:Paris_Bettencourt/ProtocolsTeam:Paris Bettencourt/Protocols2013-10-04T21:31:15Z<p>Idonnya: </p>
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<div id="page"><br />
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<img src="https://static.igem.org/mediawiki/2013/6/6c/PB_protocolosbanner.png"/><br />
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<div id="page"><br />
<h2> <b> Protocol 1:</b> Heat Shock Transformation of <i>E. coli</i></h2><br />
<p>This protocol can be used to transform chemically competent (i.e. from CaCl2) with a miniprepped plasmid or a ligation product.</p><br />
<h5>Note: Never vortex competent cells. Mix cells by gentle shaking.</h5><br />
<ol><br />
<li>Thaw competent cells on ice. These can be prepared using the CaCl2 protocol.</li><br />
<li>Place 20 ul of cells in a pre-chilled Eppendorf tube.<br />
<ul><br />
<li><u>For an Intact Vector:</u> Add 0.5 ul or less to the chilled cells</li><br />
<li><u>For a Ligation Product:</u> Add 2-3 ul to the chilled cells.</li><br />
</ul><br />
</li><br />
<li> Mix gently by flicking the tube.</li><br />
<li> Chill on ice for 10 minutes. <em>This step is optional, but can improve yields when transforming a ligation product.</em></li><br />
<li>Heat shock at 42 &deg;C for 30 seconds.</li><br />
<li>Return to ice for 2 minutes.</li><br />
<li>Add 200 ul LB medium and recover the cells by shaking at 37 &deg;C.<br /><br />
Another rich medium can substitute for the recovery.<br /><br />
The recovery time varies with the antibiotic selection.<br /><br />
Ampicillin: 15-30 minutes<br /><br />
Kanamycin or Spectinomycin: 30-60 minutes<br /><br />
Chloramphenicol: 60-120 minutes <br />
</li><br />
<li>Plate out the cells on selective LB.<br /><br />
Use glass beads to spread the cells.<br /><br />
The volume of cells plated depends on what is being transformed.<br /><br />
<ul><br />
<li><u>For an Intact Vector:</u> High transformation efficiencies are expected. Plating out 10 ul of recovered cells should produce many colonies.</li><br />
<li><u>For a Ligation Product:</u> Lower transformation efficiencies are expected. Therefore you can plate the entire 200 ul volume of recovered cells.</li><br />
</ul><br />
Note: 200 ul is the maximum volume of liquid that an LB plate can absorb.<br />
</li><br />
<li>Incubate at 37 &deg;C. Transformants should appear within 12 hrs.</li><br />
</ol><br />
<br /><br />
<br /><br />
<br />
<h2> <b> Protocol 2:</b> CaCl2 Competent Cells </h2><br />
<br />
<p>This protocol makes 4 ml of competent cells, and can be easily scaled up to make more. The cells are typically stored in 110 ul aliquots, so this will make about 35 tubes. A typical transformation uses 20 ul of cells.<br />
<br />
<h5> Note: Never vortex competent cells. Resuspend by pipetting with large Pasteur pipettes.</h5><br />
<br />
<ol><br />
<p><b><u>The night before:</u></b></p><br />
<li>The night before, inoculate a 5 ml culture and grow overnight with selection.<br />
<p><b><u>The day of:</u></b></p><br />
<li> Dilute cells ~ 1:200 into selective media.<br>For this example add 250 ul to 50 ml of selective media.<br>Note: The protocol is easily scaled to increase the number of cells.<br />
<li> Grow the cells to an OD600 of 0.6 – 0.7.<br />
<br>Use a large flask, 500ml, for good aeration.<br />
<br>Use a baffled flask for fastest growth.<br />
<br>This takes about 3 hours depending on the cells.<br />
<br>Medium-heavy cloudiness by eye is fine.<br />
<li>Spin down the cells at 4 ºC, 4000 rpm, 15 minutes.<br />
Note: Keep the cells at 4 ºC from now on.<br />
<li>Resuspend cells in 15 ml, ice-cold 100 mM CaCl2. <br />
Leave on ice 4 hours to overnight.<br />
<li>Spin down the cells at 4 ºC, 4000 rpm, 15 minutes.<br />
<li>Resuspend cells in 4 ml, ice-cold 100 mM CaCl2 + 15% glycerol.<br />
<li>Aliquot into pre-chilled Eppendorf tubes. Use immediately or store at -80ºC.<br /><br />
Note: Frozen cells are only good once.Do not refreeze cells once thawed.<br />
</ol><br />
<br /><br />
<br /><br />
</body><br />
<br />
<h2> <b> Protocol 3:</b> Glycerol Stocks </h2><br />
<br />
<ol><br />
<br />
<li>Pick Single colonies from agar plates<br />
<li>Innoculate 5ml LB broth overnight.<br />
<li>Add 750ml of overnight culture to 250ml of 60% glycerol in a cryotube.<br />
<li>Make two sets of Glycerol stocks freeze one at -20ºC and the other at -80ºC.<br />
</ol><br />
<br /><br />
<br /><br />
<br />
<h2> <b> Protocol 4: </b> Electroporation </h2><br />
<br />
<ol><br />
<b>Preparation of Electrocompetent Cells</b><br /><br />
Note: Competent cells should never be vortexted, as this will cause them to lyse <br /><br />
and release salts into the media. Resuspend cells by pipeting up and down with a large <br /><br />
pasteur pipet. Once they are chilled, cells should be continuously cold.<br /><br />
<br /><br />
<li>The night before the transformation, start an overnight culture of cells.<br /><br />
5 ml LB Amp.<br /><br />
<br /><br />
<li>The day of the transformation, dilute the cells 100X.<br /><br />
100 ml LB Amp.<br /><br />
Grow at 30&deg;C for about 90 minutes.<br /><br />
<br /><br />
<li>Harvest the cells.<br /><br />
When the cells reach an OD600 of between 0.6 and 0.8.<br /><br />
Split the culture into 2x 50 ml falcon tubes, on ice.<br /><br />
Centrifuge at 4 &deg;C for 10 min at 4000 rpm.<br /><br />
<br /><br />
<li>Wash and combine the cells.<br /><br />
Remove the supernatant.<br /><br />
Resuspend the cells in 2x 25 ml of ice cold water.<br /><br />
Combine the volumes in a single 50 ml falcon tube.<br /><br />
<br /><br />
<li>Wash the cells 2 more times.<br /><br />
Centrifuge at 4 &deg;C for 10 min at 4000 rpm.<br /><br />
Resuspend in 50 ml of ice cold water.<br /><br />
Repeat.<br /><br />
<br /><br />
<li>Wash and concentrate the cells for electroporation.<br /><br />
Centrifuge at 4 &deg;C for 10 min at 4000 rpm.<br /><br />
Resuspend in 1-2 ml of ice cold water.<br /><br />
We will use 200 ul of washed cells per transformation.<br /><br />
</ol><br />
<ol><br />
<br /><br />
<b>Dialysis of PCR or Digestion Products</b><br /><br />
Note: DNA for electroporation must be free of salts to avoid arcing.<br /><br />
<br /><br />
<li>Float a filter in a Petri dish filled with water.<br /><br />
Millipore membrane filter 0.025 uM.<br /><br />
<br /><br />
<li>Pipet one drop of PCR product onto the filter.<br /><br />
200 ng is needed per transformation.<br /><br />
20 - 100 ul fits well on one filter.<br /><br />
<br /><br />
<li>Collect the drop after 30 - 45 minutes.<br /><br />
The volume will change, but the DNA is not lost.<br />
</ol><br />
<br /><br />
<br /><br />
<br />
<h2> <b> Protocol 5:</b> Miniprep </h2><br />
<br />
<h3><b>Miniprep using <i>Thermo Scientific GeneJET Plasmid Miniprep Kit</i></b></h3><br />
<br /><br />
All centrifugations should be carried out in a table-top microcentrifuge at sup12000 x g<br /><br />
<br /><br />
<ol><br />
<li>Resuspend the pelleted cells in 250 ul of the Resuspension Solution. Transfer the cell suspension to a microcentrifuge tube. The bacteria should be resuspended completely by vortexing or pipetting up and down un<br />
til no cell clumps remain.<br />
<br /><br />
<b><i>Note</i></b>.<br />
Ensure RNase A has been added to the Resuspension Solution.<br /><br />
<br /><br />
<li>Add 250 ul of the Lysis Solution and mix thoroughly by inverting the tube 4-6 times until the solution becomes viscous and slightly clear.<br />
<br /><br />
<b><i>Note</i></b>. Do not vortex to avoid shearing of chromosomal DNA. Do not incubate for more than 5 min to avoid denaturation of supercoiled plasmid DNA.<br />
<br /><br />
<br /><br />
<li>Add 350 ul of the Neutralization Solution and mix immediately and thoroughly by inverting the tube 4-6 times.<br />
<br /><br />
<b><i>Note</i></b>.<br />
It is important to mix thoroughly and gently after the addition of the Neutralization Solution to avoid localized precipitation of bacterial cell debris. The neutralized bacterial lysate should become cloudy.<br />
<br /><br />
<br /><br />
<li>Centrifuge for 5 min to pellet cell debris and chromosomal DNA. <br />
<br /><br />
<br /><br />
<li>Transfer the supernatant to the supplied GeneJET spin column by decanting or pipetting. Avoid disturbing or transferring the white precipitate. <br />
<br /><br />
<br /><br />
<li>Centrifuge for 1 min. Discard the flow-through and place the column back into the same collection tube.<br />
<br /><br />
<b><i>Note</i></b>.<br />
Do not add bleach to the flow-through.<br />
<br /><br />
<br /><br />
<li>Add 500 ul of the Wash Solution (diluted with ethanol) to the GeneJET spin column. Centrifuge for 30-60 seconds and discard the flow-through. Place the column back into the same collection tube. <br />
<br /><br />
<br /><br />
<li>Repeat the wash procedure (step 7) using 500 ul of the Wash Solution. <br />
<br /><br />
<br /><br />
<li>Discard the flow-through and centrifuge for an additional 1 min to remove residual Wash Solution. This step is essential to avoid residual ethanol in plasmid preps. <br />
<br /><br />
<br /><br />
<li>Transfer the GeneJETspin column into a fresh 1.5 ml microcentrifuge tube. Add 50 ul of the Elution Bufferto the center of GeneJET spin column membrane to elute the plasmid DNA. Take care not to contact the membrane with the pipette tip. Incubate for 2 min at room tempera ture and centrifuge for 2 min.<br />
<br /><br />
<b><i>Note</i></b>.<br />
An additional elution step (<i>optional</i>) with Elution Buffer or water will recover residual DNA from the membrane and increase the overall yield by 10-20%. For elution of plasmids or cosmids sup20 kb, prewarm Elution Buffer to 70&deg;C before applying to silica membrane.<br />
<br /><br />
<br /><br />
<li>Discard the column and store the purified plasmid DNA at -20&deg;C. <br />
</ol><br />
<br /><br />
<br /><br />
<br />
<h2> <b> Protocol 6:</b> PCR Purification </h2><br />
<br />
<h3><b>PCR purification using <i>Thermo Scientific GeneJET PCR Purification Kit</i></b></h3><br />
<br /><br />
All centrifugations should be carried out in a table-top microcentrifuge at sup12000 x g<br /><br />
<br /><br />
<ol><br />
<li>Add a 1:1 volume of Binding Buffer to completed PCR mixture (e.g. for every 100 uL of reaction mixture, add 100 uL of Binding Buffer). Mix thoroughly. Check the color of the solution. A yellow color indicates an optimal pH for DNA binding. If the color of the solution is orange or violet, add 10 uL of 3 M sodium acetate, pH 5.2 solution and mix. The color of the mix will become yellow.<br />
<br /><br />
<br /><br />
<li><i>Optional</i>: if the DNA fragment is inf 500 bp, add a 1:2 volume of 100% isopropanol(e.g., 100 uL of isopropanol should be added to 100 uL of PCR mixture combined with 100 uL of Binding Buffer). Mix thoroughly.<br /><br />
<b><i>Note</i></b>. If PCR mixture contains primer-dimers, purification without isopropanol is recommended. However, the yield of the target DNA fragment will be lower.<br />
<br /><br />
<br /><br />
<li>Transfer up to 800 uL of the solution from step 1(or optional step 2)to the GeneJET<br />
purification column. Centrifuge for 30-60 s. Discard the flow-through.<br /><br />
<b><i>Note</i></b>. If the total volume exceeds 800 uL, the solution can be added to the column in stages. After the addition of 800 uL of solution, centrifuge the column for 30-60 s and discard flow-through. Repeat until the entire solution has been added to the column membrane.<br />
<br /><br />
<br /><br />
<li>Add 700 uL of Wash Buffer to the GeneJET purification column. Centrifuge for 30-60 s. Discard the flow-through and place the purification column back into the collection tube.<br />
<br /><br />
<br /><br />
<li>Centrifuge the empty GeneJET purification column for an additional 1 min to completely remove any residual wash buffer.<br /><br />
<b><i>Note</i></b>.This step is essential as the presence of residual ethanol in the DNA sample may inhibit subsequent reactions<br />
<br /><br />
<br /><br />
<li>Transfer the GeneJET purification column to a clean 1.5 mL microcentrifuge tube (not included).Add 50 uL of Elution Buffer to the center of the GeneJET purification column membrane and centrifuge for 1 min.<br /><br />
<b><i>Note</i></b>. For low DNA amounts the elution volumes can be reduced to increase DNA concentration. An elution volume between 20-50 uL does not significantly reduce the DNA yield. However, elution volumes less than 10 uL are not recommended. If DNA fragment is inf 10 kb, prewarm Elution Buffer to 65&deg;C before applying to column. If the elution volume is 10 uL and DNA amount is inf 5 ug, incubate column for 1 min at room temperature before centrifugation.<br />
<br /><br />
<br /><br />
<li>Discard the GeneJET purification column and store the purified DNA at -20&deg;C. <br />
</ol><br />
<br /><br />
<br /><br />
<br />
<h2> <b> Protocol 7:</b> Gel Purification </h2><br />
<br />
<h3><b>Gel purification using <i>Thermo Scientific GeneJET Gel Extraction Kit</i></b></h3><br />
<br /><br />
All centrifugations should be carried out in a table-top microcentrifuge at sup12000 x g<br /><br />
<br /><br />
<ol><br />
<li>Excise gel slice containing the DNA fragment using a clean scalpel or razor blade. Cut as close to the DNA as possible to minimize the gel volume. Place the gel slice into a pre-weighed 1.5 ml tube and weigh. Record the weight of the gel slice.<br />
<br /><br />
<b><i>Note</i></b>.<br />
If the purified fragment will be used for cloning reactions, avoid damaging the DNA through UV light exposure. Minimize UV exposure to a few seconds or keep the gel slice on a glass or plastic plate during UV illumination.<br />
<br /><br />
<br /><br />
<li>Add 1:1 volume of Binding Buffer to the gel slice (volume: weight)(e.g., add 100 ul of Binding Buffer for every 100 mg of agarose gel).<br />
<br /><br />
<b><i>Note</i></b>.<br />
For gels with an agarose content greater than 2%, a dd 2:1 volumes of Binding Buffer to the gel slice.<br />
<br /><br />
<br /><br />
<li>Incubate the gel mixture at 50-60&deg;C for 10 min or until the gel slice is completely dissolved. Mix the tube by inversion every few minutes to facilitate the melting process. Ensure that the gel is completely dissolved. Vortex the gel mixture briefly before loading on the column. Check the color of the solution. A yellow color indicates an optimal pH for DNA binding. If the color of the solution is orange or violet, add 10 ul of 3 M sodium acetate, pH 5.2 solution and mix. The color of the mix will become yellow.<br />
<br /><br />
<br /><br />
<li><i>Optional</i>: use this step only when DNA fragment is inf 500 bp or sup10 kb long. If the DNA fragment is inf 500 bp, add a 1:2 volume of 100% isopropanol to the so lubilized gel solution (e.g. 100 ul of isopropanol should be added to 100 mg gel slice solubilized in 100 ul of Binding Buffer). Mix thoroughly. If the DNA fragment is sup10 kb , add a 1:2 volume of water to the solubilized gel solution (e.g. 100 ul of water should be added to 100 mg gel slice solubilized in 100 ul of Binding Buffer). Mix thoroughly. <br />
<br /><br />
<br /><br />
<li>Transfer up to 800 ul of the solubilized gel solution (from step 3 or 4) to the GeneJET purification column. Centrifuge for 1 min. Discard the flow-through and place the column back into the same collection tube.<br />
<br /><br />
<b><i>Note</i></b>. If the total volume exceeds 800 ul, the solution can be added to the column in stages. After each application, centrifuge the column for 30-60 s and discard the flow-through aftereach spin. Repeat until the entire volume has been applied to the column membrane. Do not exceed 1 g of total agarose gel per column.<br />
<br /><br />
<br /><br />
<li><i>Optional</i>: use this additional binding step only if the purified DNA will be used for sequencing. Add 100 ul of Binding Buffer to the GeneJET purification column. Centrifuge for 1 min. Discard the flow-through and place the column back into the same collection tube.<br />
<br /><br />
<br /><br />
<li>Centrifuge the empty GeneJET purification column for an additional 1 min to completely remove residual wash buffer.<br />
<br /><br />
<b><i>Note</i></b>. This step is essential to avoid residual ethanol in the purified DNA solution. The presence of ethanol in the DNA sample may inhibit downstream enzymatic reactions.<br />
<br /><br />
<br /><br />
<li>Transfer the GeneJET purification column into a clean 1.5 ml microcentrifuge tube (not included). Add 50 ul of Elution Buffer to the center of the purification column membrane. Centrifuge for 1 min.<br />
<br /><br />
<b><i>Note</i></b>. For low DNA amounts the elution volumes can be reduced to increase DNA concentration. An elution volume between 20-50 ul does not significantly reduce the DNA yield. However, elution volumes less than 10 ul are not recommended. If DNA fragment is sup10 kb, prewarm Elution Buffer to 65&deg;C before applying to column. If the elution volume is 10 ul and DNA amount is inf5 ug, incubate column for 1 min at room temperature before centrifugation.<br />
<br /><br />
<br /><br />
<li>Discard the GeneJET purification column and store the purified DNA at -20&deg;C.<br />
</ol><br />
<br /><br />
<br /><br />
<br />
<div><br />
<h2> <b> Protocol 8: P1 Transduction</b></h2><br />
</div><br />
<div><br />
<br/><br />
</div><br />
<div><br />
This protocol uses a phage to transfer a marker from a donor strain to a recipient strain. The phage head packages about 90 kb of DNA, so donor DNA near<br />
the marker is also transferred. Note that this can cause problems if you are working with several markers that are very close together.<br />
</div><br />
<div><br />
<br/><br />
</div><br />
<div><br />
<strong><u>Lysate preparation</u></strong><br />
</div><br />
<div><br />
<br/><br />
</div><br />
<div><br />
<strong>Note: P1 phage should be stored at 4 C. It can't be frozen.</strong><br />
</div><br />
<div><br />
<br/><br />
</div><br />
<div><br />
1) The night before, start a 5 mL culture of the donor strain in selective LB.<br />
</div><br />
<div><br />
<br/><br />
</div><br />
<div><br />
2) The day of, dilute the donor strain 1:100 into Phage Lysis medium.<br />
</div><br />
<div><br />
50 ul of cells in 5 mL LB<br />
<br/><br />
</div><br />
<div><br />
+ 50 uL of 20% Glucose<br />
<br/><br />
</div><br />
<div><br />
+ 50 uL of 1M MgSO4<br />
<br/><br />
</div><br />
<div><br />
+ 25 ul of 1M CaCl2<br />
<br/><br />
</div><br />
<div><br />
No antibiotics<br />
<br/><br />
</div><br />
<div><br />
<br/><br />
</div><br />
<div><br />
3) Incubate at 37 C for 1 hour.<br />
<br/><br />
</div><br />
<div><br />
<br/><br />
</div><br />
<div><br />
4) Add 50 ul of P1 phage lysate.<br />
<br/><br />
</div><br />
<div><br />
Monitor the culture for 1-3 hours.<br />
<br/><br />
</div><br />
<div><br />
The culture should become cloudy, then clear following lysis.<br />
<br/><br />
</div><br />
<div><br />
<br/><br />
</div><br />
<div><br />
5) Add 500 ul of chloroform to the lysate and vortex.<br />
<br/><br />
</div><br />
<div><br />
<br/><br />
</div><br />
<div><br />
6) Centrifuge at max speed for 1 minute to clear the cell debris.<br />
<br/><br />
</div><br />
<div><br />
Collect the supernatant.<br />
</div><br />
<div><br />
<br/><br />
</div><br />
<div><br />
7) Phage lysate can be stored indefinitely at 4 C. Freezing will destroy the phage.<br />
</div><br />
<div><br />
</div><br />
<div><br />
<strong><u>Transduction</u></strong><br />
</div><br />
<div><br />
<br/><br />
</div><br />
<div><br />
1) The night before, start a 5 ml culture of the recipient strain in selective LB.<br />
</div><br />
<div><br />
<br/><br />
</div><br />
<div><br />
2) The day of, harvest the cells by centrifugation.<br />
<br/><br />
</div><br />
<div><br />
6000 rpm for 2 min.<br />
<br/><br />
</div><br />
<div><br />
<br/><br />
</div><br />
<div><br />
3) Resuspend in original culture volume in 5 mL Phage Infection LB.<br />
<br/><br />
</div><br />
<div><br />
5 mL LB<br />
<br/><br />
</div><br />
<div><br />
+ 50 uL of 1M MgSO4<br />
<br/><br />
</div><br />
<div><br />
+ 25 ul of 1M CaCl2<br />
<br/><br />
</div><br />
<div><br />
<br/><br />
</div><br />
<div><br />
4) Transfer 100 uL of donor P1 lysate per transformation to a 1.5 mL tube.<br />
<br/><br />
</div><br />
<div><br />
Incubate at 37 C for 30 minutes.<br />
<br/><br />
</div><br />
<div><br />
This allows the residual chloroform to evaporate.<br />
<br/><br />
</div><br />
<div><br />
<br/><br />
</div><br />
<div><br />
5) Set up 4 reactions for each transduction<br />
<br/><br />
</div><br />
<div><br />
1) 100 uL Donor Lysate 2) 10 uL Donor Lysate 3) 100 uL Donor Lysate 4) 100 uL Plain Lb<br />
<br/><br />
</div><br />
<div><br />
100 uL Recipient Cells 190 uL Recipient Cells 100 uL Plain LB 100 uL Recipient Cells<br />
</div><br />
<div><br />
<br/><br />
</div><br />
<div><br />
6) Incubate at 37 C for 30 minutes.<br />
<br/><br />
</div><br />
<div><br />
<br/><br />
</div><br />
<div><br />
7) Stop the infection with 200 uL of 1 M Sodium Citrate (pH 5.5).<br />
<br/><br />
</div><br />
<div><br />
<br/><br />
</div><br />
<div><br />
8) Add 1 mL LB and recover the cells for 1-2 hours.<br />
<br/><br />
</div><br />
<div><br />
<br/><br />
</div><br />
<div><br />
9) Spin the cells down and resuspend for plating.<br />
<br/><br />
</div><br />
<div><br />
100 ul LB + 10 uL of 1 M Sodium Citrate (pH 5.5)<br />
<br/><br />
</div><br />
<div><br />
10) Plate on selective LB.<br />
</div><br />
</ol><br />
<br /><br />
<br /><br />
<br />
<div><br />
<h2><b>Protocol 9: Colony PCR</b></h2><br />
<div><br />
<b><br />
<br/><br />
</b><br />
<div><br />
1. Pick a single colony into 30ul of nuclease-free H20. (Fresh colonies grown that day work best, but they can also come from 4C).<br />
</div><br />
<div><br />
<br/><br />
</div><br />
<div><br />
2. Boil for 10 minutes at 100C.<br />
</div><br />
<div><br />
<br/><br />
</div><br />
<div><br />
3. Centrifuge (find G) for 1 min. 1ul of this can be used directly for PCR. Best if used directly, but can also be stored at 4C for a few days.<br />
<div><br />
<p dir="ltr"><br />
<b>PCR Reaction</b><br />
</p><br />
<p dir="ltr"><br />
Keep all the reagents at 4C while preparing the mixture. Pre-heat the thermocycler to 95C and transfer your reaction directly from 4C.<br />
</p><br />
<p dir="ltr"><br />
<br/><br />
</p><br />
<div dir="ltr"><br />
<table><br />
<colgroup><br />
<col width="163"/><br />
<col width="137"/><br />
<col width="153"/><br />
<col width="143"/><br />
</colgroup><br />
<tbody><br />
<tr><br />
<td><br />
<p dir="ltr"><br />
Reagent<br />
</p><br />
</td><br />
<td><br />
<p dir="ltr"><br />
Volume ul<br />
</p><br />
</td><br />
</tr><br />
<tr><br />
<td><br />
<p dir="ltr"><br />
Forward Primer<br />
</p><br />
</td><br />
<td><br />
<p dir="ltr"><br />
1.0<br />
</p><br />
</td><br />
</tr><br />
<tr><br />
<td><br />
<p dir="ltr"><br />
Reverse Primer<br />
</p><br />
</td><br />
<td><br />
<p dir="ltr"><br />
1.0<br />
</p><br />
</td><br />
</tr><br />
<tr><br />
<td><br />
<p dir="ltr"><br />
Template DNA <br />
</p><br />
</td><br />
<td><br />
<p dir="ltr"><br />
2<br />
</p><br />
</td><br />
</tr><br />
<tr><br />
<td><br />
<p dir="ltr"><br />
Quick-Load Taq<br />
<br/><br />
2x Master Mix<br />
</p><br />
</td><br />
<td><br />
<p dir="ltr"><br />
10<br />
</p><br />
</td><br />
</tr><br />
<tr><br />
<td><br />
<p dir="ltr"><br />
Nuclease-free water<br />
</p><br />
</td><br />
<td><br />
<p dir="ltr"><br />
6<br />
</p><br />
</td><br />
</tr><br />
<tr><br />
<td><br />
<p dir="ltr"><br />
Total Volume<br />
</p><br />
</td><br />
<td><br />
<p dir="ltr"><br />
20<br />
</p><br />
</td><br />
</tr><br />
</tbody><br />
</table><br />
</div><br />
<div><br />
<br/><br />
</div><br />
<div><br />
<br/><br />
</div><br />
<div><br />
<br/><br />
<div dir="ltr"><br />
<table><br />
<colgroup><br />
<col width="148"/><br />
<col width="124"/><br />
<col width="134"/><br />
<col width="114"/><br />
<col width="104"/><br />
</colgroup><br />
<tbody><br />
<tr><br />
<td><br />
<p dir="ltr"><br />
Thermocycler Protocol: NEB Quick-Load<br />
</p><br />
</td><br />
<td><br />
<br/><br />
</td><br />
<td><br />
<br/><br />
</td><br />
<td><br />
<br/><br />
</td><br />
<td><br />
<br/><br />
</td><br />
</tr><br />
<tr><br />
<td><br />
<br/><br />
</td><br />
<td><br />
<p dir="ltr"><br />
Temp:<br />
</p><br />
</td><br />
<td><br />
<p dir="ltr"><br />
Time<br />
</p><br />
</td><br />
<td><br />
<br/><br />
</td><br />
<td><br />
<br/><br />
</td><br />
</tr><br />
<tr><br />
<td><br />
<p dir="ltr"><br />
Start<br />
</p><br />
</td><br />
<td><br />
<p dir="ltr"><br />
95C<br />
</p><br />
</td><br />
<td><br />
<p dir="ltr"><br />
30sec<br />
</p><br />
</td><br />
<td><br />
<p dir="ltr"><br />
Melt<br />
</p><br />
</td><br />
<td><br />
<br/><br />
</td><br />
</tr><br />
<tr><br />
<td><br />
<br/><br />
</td><br />
<td><br />
<br/><br />
</td><br />
<td><br />
<br/><br />
</td><br />
<td><br />
<br/><br />
</td><br />
<td><br />
<br/><br />
</td><br />
</tr><br />
<tr><br />
<td><br />
<p dir="ltr"><br />
Cycle 1<br />
</p><br />
</td><br />
<td><br />
<p dir="ltr"><br />
95C<br />
</p><br />
</td><br />
<td><br />
<p dir="ltr"><br />
15sec<br />
</p><br />
</td><br />
<td><br />
<p dir="ltr"><br />
Melt<br />
</p><br />
</td><br />
<td><br />
<p dir="ltr"><br />
30 Cycles<br />
</p><br />
</td><br />
</tr><br />
<tr><br />
<td><br />
<p dir="ltr"><br />
Cycle 2<br />
</p><br />
</td><br />
<td><br />
<p dir="ltr"><br />
60C<br />
</p><br />
</td><br />
<td><br />
<p dir="ltr"><br />
30sec<br />
</p><br />
</td><br />
<td><br />
<p dir="ltr"><br />
Anneal<br />
</p><br />
</td><br />
<td><br />
<p dir="ltr"><br />
30 Cycles<br />
</p><br />
</td><br />
</tr><br />
<tr><br />
<td><br />
<p dir="ltr"><br />
Cycle 3<br />
</p><br />
</td><br />
<td><br />
<p dir="ltr"><br />
72C<br />
</p><br />
</td><br />
<td><br />
<p dir="ltr"><br />
1 min per kb<br />
</p><br />
</td><br />
<td><br />
<p dir="ltr"><br />
Extend<br />
</p><br />
</td><br />
<td><br />
<p dir="ltr"><br />
30 Cycles<br />
</p><br />
</td><br />
</tr><br />
<tr><br />
<td><br />
<br/><br />
</td><br />
<td><br />
<br/><br />
</td><br />
<td><br />
<br/><br />
</td><br />
<td><br />
<br/><br />
</td><br />
<td><br />
<br/><br />
</td><br />
</tr><br />
<tr><br />
<td><br />
<p dir="ltr"><br />
Finish<br />
</p><br />
</td><br />
<td><br />
<p dir="ltr"><br />
72C<br />
</p><br />
</td><br />
<td><br />
<p dir="ltr"><br />
5 min<br />
</p><br />
</td><br />
<td><br />
<p dir="ltr"><br />
Extend<br />
</p><br />
</td><br />
<td><br />
<br/><br />
</td><br />
</tr><br />
<tr><br />
<td><br />
<p dir="ltr"><br />
Store<br />
</p><br />
</td><br />
<td><br />
<p dir="ltr"><br />
10C<br />
</p><br />
</td><br />
<td><br />
<p dir="ltr"><br />
Forever<br />
</p><br />
</td><br />
<td><br />
<p dir="ltr"><br />
Store<br />
</p><br />
</td><br />
<td><br />
<br/><br />
</td><br />
</tr><br />
</tbody><br />
</table><br />
</div><br />
<div dir="ltr"><br />
<p dir="ltr"><br />
The annealing temperature may vary from 45-68C, depending mostly on your primer.<br />
</p><br />
Samples can then be used for gel electrophoresis.<br />
<br/><br />
</div><br />
</div><br />
</div><br />
</div><br />
</div><br />
</ol><br />
<br /><br />
<br /><br />
<br />
<h2><b>Protocol 10: Preparation of LB liquid broth:</b></h2><br />
</div><br />
<p dir="ltr"><br />
LB is our standard rich media. Directions are on the bottle.<br />
</p><br />
<p dir="ltr"><br />
1. Add 12.5g of LB broth powder to a 1L glass bottle.<br />
</p><br />
<p dir="ltr"><br />
2. Add 500ml of osmotic water.<br />
</p><br />
<p dir="ltr"><br />
3. Autoclave <br />
</p><br />
<p dir="ltr"><br />
<br/><br />
</p><br />
<div dir="ltr"><br />
<br/><br />
</div><br />
</ol><br />
<br /><br />
<br /><br />
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</div><br />
</html><br />
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{{:Team:Paris_Bettencourt/footer}}</div>Idonnyahttp://2013.igem.org/Team:Paris_Bettencourt/ProtocolsTeam:Paris Bettencourt/Protocols2013-10-04T21:24:35Z<p>Idonnya: </p>
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<h2> <b> Protocol 1:</b> Heat Shock Transformation of <i>E. coli</i></h2><br />
<p>This protocol can be used to transform chemically competent (i.e. from CaCl2) with a miniprepped plasmid or a ligation product.</p><br />
<h5>Note: Never vortex competent cells. Mix cells by gentle shaking.</h5><br />
<ol><br />
<li>Thaw competent cells on ice. These can be prepared using the CaCl2 protocol.</li><br />
<li>Place 20 ul of cells in a pre-chilled Eppendorf tube.<br />
<ul><br />
<li><u>For an Intact Vector:</u> Add 0.5 ul or less to the chilled cells</li><br />
<li><u>For a Ligation Product:</u> Add 2-3 ul to the chilled cells.</li><br />
</ul><br />
</li><br />
<li> Mix gently by flicking the tube.</li><br />
<li> Chill on ice for 10 minutes. <em>This step is optional, but can improve yields when transforming a ligation product.</em></li><br />
<li>Heat shock at 42 &deg;C for 30 seconds.</li><br />
<li>Return to ice for 2 minutes.</li><br />
<li>Add 200 ul LB medium and recover the cells by shaking at 37 &deg;C.<br /><br />
Another rich medium can substitute for the recovery.<br /><br />
The recovery time varies with the antibiotic selection.<br /><br />
Ampicillin: 15-30 minutes<br /><br />
Kanamycin or Spectinomycin: 30-60 minutes<br /><br />
Chloramphenicol: 60-120 minutes <br />
</li><br />
<li>Plate out the cells on selective LB.<br /><br />
Use glass beads to spread the cells.<br /><br />
The volume of cells plated depends on what is being transformed.<br /><br />
<ul><br />
<li><u>For an Intact Vector:</u> High transformation efficiencies are expected. Plating out 10 ul of recovered cells should produce many colonies.</li><br />
<li><u>For a Ligation Product:</u> Lower transformation efficiencies are expected. Therefore you can plate the entire 200 ul volume of recovered cells.</li><br />
</ul><br />
Note: 200 ul is the maximum volume of liquid that an LB plate can absorb.<br />
</li><br />
<li>Incubate at 37 &deg;C. Transformants should appear within 12 hrs.</li><br />
</ol><br />
<br /><br />
<br /><br />
<br />
<h2> <b> Protocol 2:</b> CaCl2 Competent Cells </h2><br />
<br />
<p>This protocol makes 4 ml of competent cells, and can be easily scaled up to make more. The cells are typically stored in 110 ul aliquots, so this will make about 35 tubes. A typical transformation uses 20 ul of cells.<br />
<br />
<h5> Note: Never vortex competent cells. Resuspend by pipetting with large Pasteur pipettes.</h5><br />
<br />
<ol><br />
<p><b><u>The night before:</u></b></p><br />
<li>The night before, inoculate a 5 ml culture and grow overnight with selection.<br />
<p><b><u>The day of:</u></b></p><br />
<li> Dilute cells ~ 1:200 into selective media.<br>For this example add 250 ul to 50 ml of selective media.<br>Note: The protocol is easily scaled to increase the number of cells.<br />
<li> Grow the cells to an OD600 of 0.6 – 0.7.<br />
<br>Use a large flask, 500ml, for good aeration.<br />
<br>Use a baffled flask for fastest growth.<br />
<br>This takes about 3 hours depending on the cells.<br />
<br>Medium-heavy cloudiness by eye is fine.<br />
<li>Spin down the cells at 4 ºC, 4000 rpm, 15 minutes.<br />
Note: Keep the cells at 4 ºC from now on.<br />
<li>Resuspend cells in 15 ml, ice-cold 100 mM CaCl2. <br />
Leave on ice 4 hours to overnight.<br />
<li>Spin down the cells at 4 ºC, 4000 rpm, 15 minutes.<br />
<li>Resuspend cells in 4 ml, ice-cold 100 mM CaCl2 + 15% glycerol.<br />
<li>Aliquot into pre-chilled Eppendorf tubes. Use immediately or store at -80ºC.<br /><br />
Note: Frozen cells are only good once.Do not refreeze cells once thawed.<br />
</ol><br />
<br /><br />
<br /><br />
</body><br />
<br />
<h2> <b> Protocol 3:</b> Glycerol Stocks </h2><br />
<br />
<ol><br />
<br />
<li>Pick Single colonies from agar plates<br />
<li>Innoculate 5ml LB broth overnight.<br />
<li>Add 750ml of overnight culture to 250ml of 60% glycerol in a cryotube.<br />
<li>Make two sets of Glycerol stocks freeze one at -20ºC and the other at -80ºC.<br />
</ol><br />
<br /><br />
<br /><br />
<br />
<h2> <b> Protocol 4: </b> Electroporation </h2><br />
<br />
<ol><br />
<b>Preparation of Electrocompetent Cells</b><br /><br />
Note: Competent cells should never be vortexted, as this will cause them to lyse <br /><br />
and release salts into the media. Resuspend cells by pipeting up and down with a large <br /><br />
pasteur pipet. Once they are chilled, cells should be continuously cold.<br /><br />
<br /><br />
<li>The night before the transformation, start an overnight culture of cells.<br /><br />
5 ml LB Amp.<br /><br />
<br /><br />
<li>The day of the transformation, dilute the cells 100X.<br /><br />
100 ml LB Amp.<br /><br />
Grow at 30&deg;C for about 90 minutes.<br /><br />
<br /><br />
<li>Harvest the cells.<br /><br />
When the cells reach an OD600 of between 0.6 and 0.8.<br /><br />
Split the culture into 2x 50 ml falcon tubes, on ice.<br /><br />
Centrifuge at 4 &deg;C for 10 min at 4000 rpm.<br /><br />
<br /><br />
<li>Wash and combine the cells.<br /><br />
Remove the supernatant.<br /><br />
Resuspend the cells in 2x 25 ml of ice cold water.<br /><br />
Combine the volumes in a single 50 ml falcon tube.<br /><br />
<br /><br />
<li>Wash the cells 2 more times.<br /><br />
Centrifuge at 4 &deg;C for 10 min at 4000 rpm.<br /><br />
Resuspend in 50 ml of ice cold water.<br /><br />
Repeat.<br /><br />
<br /><br />
<li>Wash and concentrate the cells for electroporation.<br /><br />
Centrifuge at 4 &deg;C for 10 min at 4000 rpm.<br /><br />
Resuspend in 1-2 ml of ice cold water.<br /><br />
We will use 200 ul of washed cells per transformation.<br /><br />
</ol><br />
<ol><br />
<br /><br />
<b>Dialysis of PCR or Digestion Products</b><br /><br />
Note: DNA for electroporation must be free of salts to avoid arcing.<br /><br />
<br /><br />
<li>Float a filter in a Petri dish filled with water.<br /><br />
Millipore membrane filter 0.025 uM.<br /><br />
<br /><br />
<li>Pipet one drop of PCR product onto the filter.<br /><br />
200 ng is needed per transformation.<br /><br />
20 - 100 ul fits well on one filter.<br /><br />
<br /><br />
<li>Collect the drop after 30 - 45 minutes.<br /><br />
The volume will change, but the DNA is not lost.<br />
</ol><br />
<br /><br />
<br /><br />
<br />
<h2> <b> Protocol 5:</b> Miniprep </h2><br />
<br />
<h3><b>Miniprep using <i>Thermo Scientific GeneJET Plasmid Miniprep Kit</i></b></h3><br />
<br /><br />
All centrifugations should be carried out in a table-top microcentrifuge at sup12000 x g<br /><br />
<br /><br />
<ol><br />
<li>Resuspend the pelleted cells in 250 ul of the Resuspension Solution. Transfer the cell suspension to a microcentrifuge tube. The bacteria should be resuspended completely by vortexing or pipetting up and down un<br />
til no cell clumps remain.<br />
<br /><br />
<b><i>Note</i></b>.<br />
Ensure RNase A has been added to the Resuspension Solution.<br /><br />
<br /><br />
<li>Add 250 ul of the Lysis Solution and mix thoroughly by inverting the tube 4-6 times until the solution becomes viscous and slightly clear.<br />
<br /><br />
<b><i>Note</i></b>. Do not vortex to avoid shearing of chromosomal DNA. Do not incubate for more than 5 min to avoid denaturation of supercoiled plasmid DNA.<br />
<br /><br />
<br /><br />
<li>Add 350 ul of the Neutralization Solution and mix immediately and thoroughly by inverting the tube 4-6 times.<br />
<br /><br />
<b><i>Note</i></b>.<br />
It is important to mix thoroughly and gently after the addition of the Neutralization Solution to avoid localized precipitation of bacterial cell debris. The neutralized bacterial lysate should become cloudy.<br />
<br /><br />
<br /><br />
<li>Centrifuge for 5 min to pellet cell debris and chromosomal DNA. <br />
<br /><br />
<br /><br />
<li>Transfer the supernatant to the supplied GeneJET spin column by decanting or pipetting. Avoid disturbing or transferring the white precipitate. <br />
<br /><br />
<br /><br />
<li>Centrifuge for 1 min. Discard the flow-through and place the column back into the same collection tube.<br />
<br /><br />
<b><i>Note</i></b>.<br />
Do not add bleach to the flow-through.<br />
<br /><br />
<br /><br />
<li>Add 500 ul of the Wash Solution (diluted with ethanol) to the GeneJET spin column. Centrifuge for 30-60 seconds and discard the flow-through. Place the column back into the same collection tube. <br />
<br /><br />
<br /><br />
<li>Repeat the wash procedure (step 7) using 500 ul of the Wash Solution. <br />
<br /><br />
<br /><br />
<li>Discard the flow-through and centrifuge for an additional 1 min to remove residual Wash Solution. This step is essential to avoid residual ethanol in plasmid preps. <br />
<br /><br />
<br /><br />
<li>Transfer the GeneJETspin column into a fresh 1.5 ml microcentrifuge tube. Add 50 ul of the Elution Bufferto the center of GeneJET spin column membrane to elute the plasmid DNA. Take care not to contact the membrane with the pipette tip. Incubate for 2 min at room tempera ture and centrifuge for 2 min.<br />
<br /><br />
<b><i>Note</i></b>.<br />
An additional elution step (<i>optional</i>) with Elution Buffer or water will recover residual DNA from the membrane and increase the overall yield by 10-20%. For elution of plasmids or cosmids sup20 kb, prewarm Elution Buffer to 70&deg;C before applying to silica membrane.<br />
<br /><br />
<br /><br />
<li>Discard the column and store the purified plasmid DNA at -20&deg;C. <br />
</ol><br />
<br /><br />
<br /><br />
<br />
<h2> <b> Protocol 6:</b> PCR Purification </h2><br />
<br />
<h3><b>PCR purification using <i>Thermo Scientific GeneJET PCR Purification Kit</i></b></h3><br />
<br /><br />
All centrifugations should be carried out in a table-top microcentrifuge at sup12000 x g<br /><br />
<br /><br />
<ol><br />
<li>Add a 1:1 volume of Binding Buffer to completed PCR mixture (e.g. for every 100 uL of reaction mixture, add 100 uL of Binding Buffer). Mix thoroughly. Check the color of the solution. A yellow color indicates an optimal pH for DNA binding. If the color of the solution is orange or violet, add 10 uL of 3 M sodium acetate, pH 5.2 solution and mix. The color of the mix will become yellow.<br />
<br /><br />
<br /><br />
<li><i>Optional</i>: if the DNA fragment is inf 500 bp, add a 1:2 volume of 100% isopropanol(e.g., 100 uL of isopropanol should be added to 100 uL of PCR mixture combined with 100 uL of Binding Buffer). Mix thoroughly.<br /><br />
<b><i>Note</i></b>. If PCR mixture contains primer-dimers, purification without isopropanol is recommended. However, the yield of the target DNA fragment will be lower.<br />
<br /><br />
<br /><br />
<li>Transfer up to 800 uL of the solution from step 1(or optional step 2)to the GeneJET<br />
purification column. Centrifuge for 30-60 s. Discard the flow-through.<br /><br />
<b><i>Note</i></b>. If the total volume exceeds 800 uL, the solution can be added to the column in stages. After the addition of 800 uL of solution, centrifuge the column for 30-60 s and discard flow-through. Repeat until the entire solution has been added to the column membrane.<br />
<br /><br />
<br /><br />
<li>Add 700 uL of Wash Buffer to the GeneJET purification column. Centrifuge for 30-60 s. Discard the flow-through and place the purification column back into the collection tube.<br />
<br /><br />
<br /><br />
<li>Centrifuge the empty GeneJET purification column for an additional 1 min to completely remove any residual wash buffer.<br /><br />
<b><i>Note</i></b>.This step is essential as the presence of residual ethanol in the DNA sample may inhibit subsequent reactions<br />
<br /><br />
<br /><br />
<li>Transfer the GeneJET purification column to a clean 1.5 mL microcentrifuge tube (not included).Add 50 uL of Elution Buffer to the center of the GeneJET purification column membrane and centrifuge for 1 min.<br /><br />
<b><i>Note</i></b>. For low DNA amounts the elution volumes can be reduced to increase DNA concentration. An elution volume between 20-50 uL does not significantly reduce the DNA yield. However, elution volumes less than 10 uL are not recommended. If DNA fragment is inf 10 kb, prewarm Elution Buffer to 65&deg;C before applying to column. If the elution volume is 10 uL and DNA amount is inf 5 ug, incubate column for 1 min at room temperature before centrifugation.<br />
<br /><br />
<br /><br />
<li>Discard the GeneJET purification column and store the purified DNA at -20&deg;C. <br />
</ol><br />
<br /><br />
<br /><br />
<br />
<h2> <b> Protocol 7:</b> Gel Purification </h2><br />
<br />
<h3><b>Gel purification using <i>Thermo Scientific GeneJET Gel Extraction Kit</i></b></h3><br />
<br /><br />
All centrifugations should be carried out in a table-top microcentrifuge at sup12000 x g<br /><br />
<br /><br />
<ol><br />
<li>Excise gel slice containing the DNA fragment using a clean scalpel or razor blade. Cut as close to the DNA as possible to minimize the gel volume. Place the gel slice into a pre-weighed 1.5 ml tube and weigh. Record the weight of the gel slice.<br />
<br /><br />
<b><i>Note</i></b>.<br />
If the purified fragment will be used for cloning reactions, avoid damaging the DNA through UV light exposure. Minimize UV exposure to a few seconds or keep the gel slice on a glass or plastic plate during UV illumination.<br />
<br /><br />
<br /><br />
<li>Add 1:1 volume of Binding Buffer to the gel slice (volume: weight)(e.g., add 100 ul of Binding Buffer for every 100 mg of agarose gel).<br />
<br /><br />
<b><i>Note</i></b>.<br />
For gels with an agarose content greater than 2%, a dd 2:1 volumes of Binding Buffer to the gel slice.<br />
<br /><br />
<br /><br />
<li>Incubate the gel mixture at 50-60&deg;C for 10 min or until the gel slice is completely dissolved. Mix the tube by inversion every few minutes to facilitate the melting process. Ensure that the gel is completely dissolved. Vortex the gel mixture briefly before loading on the column. Check the color of the solution. A yellow color indicates an optimal pH for DNA binding. If the color of the solution is orange or violet, add 10 ul of 3 M sodium acetate, pH 5.2 solution and mix. The color of the mix will become yellow.<br />
<br /><br />
<br /><br />
<li><i>Optional</i>: use this step only when DNA fragment is inf 500 bp or sup10 kb long. If the DNA fragment is inf 500 bp, add a 1:2 volume of 100% isopropanol to the so lubilized gel solution (e.g. 100 ul of isopropanol should be added to 100 mg gel slice solubilized in 100 ul of Binding Buffer). Mix thoroughly. If the DNA fragment is sup10 kb , add a 1:2 volume of water to the solubilized gel solution (e.g. 100 ul of water should be added to 100 mg gel slice solubilized in 100 ul of Binding Buffer). Mix thoroughly. <br />
<br /><br />
<br /><br />
<li>Transfer up to 800 ul of the solubilized gel solution (from step 3 or 4) to the GeneJET purification column. Centrifuge for 1 min. Discard the flow-through and place the column back into the same collection tube.<br />
<br /><br />
<b><i>Note</i></b>. If the total volume exceeds 800 ul, the solution can be added to the column in stages. After each application, centrifuge the column for 30-60 s and discard the flow-through aftereach spin. Repeat until the entire volume has been applied to the column membrane. Do not exceed 1 g of total agarose gel per column.<br />
<br /><br />
<br /><br />
<li><i>Optional</i>: use this additional binding step only if the purified DNA will be used for sequencing. Add 100 ul of Binding Buffer to the GeneJET purification column. Centrifuge for 1 min. Discard the flow-through and place the column back into the same collection tube.<br />
<br /><br />
<br /><br />
<li>Centrifuge the empty GeneJET purification column for an additional 1 min to completely remove residual wash buffer.<br />
<br /><br />
<b><i>Note</i></b>. This step is essential to avoid residual ethanol in the purified DNA solution. The presence of ethanol in the DNA sample may inhibit downstream enzymatic reactions.<br />
<br /><br />
<br /><br />
<li>Transfer the GeneJET purification column into a clean 1.5 ml microcentrifuge tube (not included). Add 50 ul of Elution Buffer to the center of the purification column membrane. Centrifuge for 1 min.<br />
<br /><br />
<b><i>Note</i></b>. For low DNA amounts the elution volumes can be reduced to increase DNA concentration. An elution volume between 20-50 ul does not significantly reduce the DNA yield. However, elution volumes less than 10 ul are not recommended. If DNA fragment is sup10 kb, prewarm Elution Buffer to 65&deg;C before applying to column. If the elution volume is 10 ul and DNA amount is inf5 ug, incubate column for 1 min at room temperature before centrifugation.<br />
<br /><br />
<br /><br />
<li>Discard the GeneJET purification column and store the purified DNA at -20&deg;C.<br />
</ol><br />
<br /><br />
<br /><br />
<br />
<div><br />
<h2> <b> Protocol 8: P1 Transduction</b></h2><br />
</div><br />
<div><br />
<br/><br />
</div><br />
<div><br />
This protocol uses a phage to transfer a marker from a donor strain to a recipient strain. The phage head packages about 90 kb of DNA, so donor DNA near<br />
the marker is also transferred. Note that this can cause problems if you are working with several markers that are very close together.<br />
</div><br />
<div><br />
<br/><br />
</div><br />
<div><br />
This protocol uses a phage to transfer a marker from a donor strain to a recipient strain. The phage head packages about 90 kb of DNA, so donor DNA near<br />
the marker is also transferred. Note that this can cause problems if you are working with several markers that are very close together.<br />
</div><br />
<div><br />
<br/><br />
</div><br />
<div><br />
<strong><u>Lysate preparation</u></strong><br />
</div><br />
<div><br />
<br/><br />
</div><br />
<div><br />
<strong>Note: P1 phage should be stored at 4 C. It can't be frozen.</strong><br />
</div><br />
<div><br />
<br/><br />
</div><br />
<div><br />
1) The night before, start a 5 mL culture of the donor strain in selective LB.<br />
</div><br />
<div><br />
<br/><br />
</div><br />
<div><br />
2) The day of, dilute the donor strain 1:100 into Phage Lysis medium.<br />
</div><br />
<div><br />
50 ul of cells in 5 mL LB<br />
<br/><br />
</div><br />
<div><br />
+ 50 uL of 20% Glucose<br />
<br/><br />
</div><br />
<div><br />
+ 50 uL of 1M MgSO4<br />
<br/><br />
</div><br />
<div><br />
+ 25 ul of 1M CaCl2<br />
<br/><br />
</div><br />
<div><br />
No antibiotics<br />
<br/><br />
</div><br />
<div><br />
<br/><br />
</div><br />
<div><br />
3) Incubate at 37 C for 1 hour.<br />
<br/><br />
</div><br />
<div><br />
<br/><br />
</div><br />
<div><br />
4) Add 50 ul of P1 phage lysate.<br />
<br/><br />
</div><br />
<div><br />
Monitor the culture for 1-3 hours.<br />
<br/><br />
</div><br />
<div><br />
The culture should become cloudy, then clear following lysis.<br />
<br/><br />
</div><br />
<div><br />
<br/><br />
</div><br />
<div><br />
5) Add 500 ul of chloroform to the lysate and vortex.<br />
<br/><br />
</div><br />
<div><br />
<br/><br />
</div><br />
<div><br />
6) Centrifuge at max speed for 1 minute to clear the cell debris.<br />
<br/><br />
</div><br />
<div><br />
Collect the supernatant.<br />
</div><br />
<div><br />
<br/><br />
</div><br />
<div><br />
7) Phage lysate can be stored indefinitely at 4 C. Freezing will destroy the phage.<br />
</div><br />
<div><br />
</div><br />
<div><br />
<strong><u>Transduction</u></strong><br />
</div><br />
<div><br />
<br/><br />
</div><br />
<div><br />
1) The night before, start a 5 ml culture of the recipient strain in selective LB.<br />
</div><br />
<div><br />
<br/><br />
</div><br />
<div><br />
2) The day of, harvest the cells by centrifugation.<br />
<br/><br />
</div><br />
<div><br />
6000 rpm for 2 min.<br />
<br/><br />
</div><br />
<div><br />
<br/><br />
</div><br />
<div><br />
3) Resuspend in original culture volume in 5 mL Phage Infection LB.<br />
<br/><br />
</div><br />
<div><br />
5 mL LB<br />
<br/><br />
</div><br />
<div><br />
+ 50 uL of 1M MgSO4<br />
<br/><br />
</div><br />
<div><br />
+ 25 ul of 1M CaCl2<br />
<br/><br />
</div><br />
<div><br />
<br/><br />
</div><br />
<div><br />
4) Transfer 100 uL of donor P1 lysate per transformation to a 1.5 mL tube.<br />
<br/><br />
</div><br />
<div><br />
Incubate at 37 C for 30 minutes.<br />
<br/><br />
</div><br />
<div><br />
This allows the residual chloroform to evaporate.<br />
<br/><br />
</div><br />
<div><br />
<br/><br />
</div><br />
<div><br />
5) Set up 4 reactions for each transduction<br />
<br/><br />
</div><br />
<div><br />
1) 100 uL Donor Lysate 2) 10 uL Donor Lysate 3) 100 uL Donor Lysate 4) 100 uL Plain Lb<br />
<br/><br />
</div><br />
<div><br />
100 uL Recipient Cells 190 uL Recipient Cells 100 uL Plain LB 100 uL Recipient Cells<br />
</div><br />
<div><br />
<br/><br />
</div><br />
<div><br />
6) Incubate at 37 C for 30 minutes.<br />
<br/><br />
</div><br />
<div><br />
<br/><br />
</div><br />
<div><br />
7) Stop the infection with 200 uL of 1 M Sodium Citrate (pH 5.5).<br />
<br/><br />
</div><br />
<div><br />
<br/><br />
</div><br />
<div><br />
8) Add 1 mL LB and recover the cells for 1-2 hours.<br />
<br/><br />
</div><br />
<div><br />
<br/><br />
</div><br />
<div><br />
9) Spin the cells down and resuspend for plating.<br />
<br/><br />
</div><br />
<div><br />
100 ul LB + 10 uL of 1 M Sodium Citrate (pH 5.5)<br />
<br/><br />
</div><br />
<div><br />
10) Plate on selective LB.<br />
</div><br />
</ol><br />
<br /><br />
<br /><br />
<br />
<div><br />
<h2><b>Protocol 9: Colony PCR</b></h2><br />
<div><br />
<b><br />
<br/><br />
</b><br />
<div><br />
1. Pick a single colony into 30ul of nuclease-free H20. (Fresh colonies grown that day work best, but they can also come from 4C).<br />
</div><br />
<div><br />
<br/><br />
</div><br />
<div><br />
2. Boil for 10 minutes at 100C.<br />
</div><br />
<div><br />
<br/><br />
</div><br />
<div><br />
3. Centrifuge (find G) for 1 min. 1ul of this can be used directly for PCR. Best if used directly, but can also be stored at 4C for a few days.<br />
<div><br />
<p dir="ltr"><br />
<b>PCR Reaction</b><br />
</p><br />
<p dir="ltr"><br />
Keep all the reagents at 4C while preparing the mixture. Pre-heat the thermocycler to 95C and transfer your reaction directly from 4C.<br />
</p><br />
<p dir="ltr"><br />
<br/><br />
</p><br />
<div dir="ltr"><br />
<table><br />
<colgroup><br />
<col width="163"/><br />
<col width="137"/><br />
<col width="153"/><br />
<col width="143"/><br />
</colgroup><br />
<tbody><br />
<tr><br />
<td><br />
<p dir="ltr"><br />
Reagent<br />
</p><br />
</td><br />
<td><br />
<p dir="ltr"><br />
Volume ul<br />
</p><br />
</td><br />
</tr><br />
<tr><br />
<td><br />
<p dir="ltr"><br />
Forward Primer<br />
</p><br />
</td><br />
<td><br />
<p dir="ltr"><br />
1.0<br />
</p><br />
</td><br />
</tr><br />
<tr><br />
<td><br />
<p dir="ltr"><br />
Reverse Primer<br />
</p><br />
</td><br />
<td><br />
<p dir="ltr"><br />
1.0<br />
</p><br />
</td><br />
</tr><br />
<tr><br />
<td><br />
<p dir="ltr"><br />
Template DNA <br />
</p><br />
</td><br />
<td><br />
<p dir="ltr"><br />
2<br />
</p><br />
</td><br />
</tr><br />
<tr><br />
<td><br />
<p dir="ltr"><br />
Quick-Load Taq<br />
<br/><br />
2x Master Mix<br />
</p><br />
</td><br />
<td><br />
<p dir="ltr"><br />
10<br />
</p><br />
</td><br />
</tr><br />
<tr><br />
<td><br />
<p dir="ltr"><br />
Nuclease-free water<br />
</p><br />
</td><br />
<td><br />
<p dir="ltr"><br />
6<br />
</p><br />
</td><br />
</tr><br />
<tr><br />
<td><br />
<p dir="ltr"><br />
Total Volume<br />
</p><br />
</td><br />
<td><br />
<p dir="ltr"><br />
20<br />
</p><br />
</td><br />
</tr><br />
</tbody><br />
</table><br />
</div><br />
<div><br />
<br/><br />
</div><br />
<div><br />
<br/><br />
</div><br />
<div><br />
<br/><br />
<div dir="ltr"><br />
<table><br />
<colgroup><br />
<col width="148"/><br />
<col width="124"/><br />
<col width="134"/><br />
<col width="114"/><br />
<col width="104"/><br />
</colgroup><br />
<tbody><br />
<tr><br />
<td><br />
<p dir="ltr"><br />
Thermocycler Protocol: NEB Quick-Load<br />
</p><br />
</td><br />
<td><br />
<br/><br />
</td><br />
<td><br />
<br/><br />
</td><br />
<td><br />
<br/><br />
</td><br />
<td><br />
<br/><br />
</td><br />
</tr><br />
<tr><br />
<td><br />
<br/><br />
</td><br />
<td><br />
<p dir="ltr"><br />
Temp:<br />
</p><br />
</td><br />
<td><br />
<p dir="ltr"><br />
Time<br />
</p><br />
</td><br />
<td><br />
<br/><br />
</td><br />
<td><br />
<br/><br />
</td><br />
</tr><br />
<tr><br />
<td><br />
<p dir="ltr"><br />
Start<br />
</p><br />
</td><br />
<td><br />
<p dir="ltr"><br />
95C<br />
</p><br />
</td><br />
<td><br />
<p dir="ltr"><br />
30sec<br />
</p><br />
</td><br />
<td><br />
<p dir="ltr"><br />
Melt<br />
</p><br />
</td><br />
<td><br />
<br/><br />
</td><br />
</tr><br />
<tr><br />
<td><br />
<br/><br />
</td><br />
<td><br />
<br/><br />
</td><br />
<td><br />
<br/><br />
</td><br />
<td><br />
<br/><br />
</td><br />
<td><br />
<br/><br />
</td><br />
</tr><br />
<tr><br />
<td><br />
<p dir="ltr"><br />
Cycle 1<br />
</p><br />
</td><br />
<td><br />
<p dir="ltr"><br />
95C<br />
</p><br />
</td><br />
<td><br />
<p dir="ltr"><br />
15sec<br />
</p><br />
</td><br />
<td><br />
<p dir="ltr"><br />
Melt<br />
</p><br />
</td><br />
<td><br />
<p dir="ltr"><br />
30 Cycles<br />
</p><br />
</td><br />
</tr><br />
<tr><br />
<td><br />
<p dir="ltr"><br />
Cycle 2<br />
</p><br />
</td><br />
<td><br />
<p dir="ltr"><br />
60C<br />
</p><br />
</td><br />
<td><br />
<p dir="ltr"><br />
30sec<br />
</p><br />
</td><br />
<td><br />
<p dir="ltr"><br />
Anneal<br />
</p><br />
</td><br />
<td><br />
<p dir="ltr"><br />
30 Cycles<br />
</p><br />
</td><br />
</tr><br />
<tr><br />
<td><br />
<p dir="ltr"><br />
Cycle 3<br />
</p><br />
</td><br />
<td><br />
<p dir="ltr"><br />
72C<br />
</p><br />
</td><br />
<td><br />
<p dir="ltr"><br />
1 min per kb<br />
</p><br />
</td><br />
<td><br />
<p dir="ltr"><br />
Extend<br />
</p><br />
</td><br />
<td><br />
<p dir="ltr"><br />
30 Cycles<br />
</p><br />
</td><br />
</tr><br />
<tr><br />
<td><br />
<br/><br />
</td><br />
<td><br />
<br/><br />
</td><br />
<td><br />
<br/><br />
</td><br />
<td><br />
<br/><br />
</td><br />
<td><br />
<br/><br />
</td><br />
</tr><br />
<tr><br />
<td><br />
<p dir="ltr"><br />
Finish<br />
</p><br />
</td><br />
<td><br />
<p dir="ltr"><br />
72C<br />
</p><br />
</td><br />
<td><br />
<p dir="ltr"><br />
5 min<br />
</p><br />
</td><br />
<td><br />
<p dir="ltr"><br />
Extend<br />
</p><br />
</td><br />
<td><br />
<br/><br />
</td><br />
</tr><br />
<tr><br />
<td><br />
<p dir="ltr"><br />
Store<br />
</p><br />
</td><br />
<td><br />
<p dir="ltr"><br />
10C<br />
</p><br />
</td><br />
<td><br />
<p dir="ltr"><br />
Forever<br />
</p><br />
</td><br />
<td><br />
<p dir="ltr"><br />
Store<br />
</p><br />
</td><br />
<td><br />
<br/><br />
</td><br />
</tr><br />
</tbody><br />
</table><br />
</div><br />
<div dir="ltr"><br />
<p dir="ltr"><br />
The annealing temperature may vary from 45-68C, depending mostly on your primer.<br />
</p><br />
Samples can then be used for gel electrophoresis.<br />
<br/><br />
</div><br />
</div><br />
</div><br />
</div><br />
</div><br />
</ol><br />
<br /><br />
<br /><br />
<br />
<br />
</div><br />
</html><br />
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{{:Team:Paris_Bettencourt/footer}}</div>Idonnyahttp://2013.igem.org/Team:Paris_Bettencourt/ProtocolsTeam:Paris Bettencourt/Protocols2013-10-04T20:25:05Z<p>Idonnya: </p>
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<h2> <b> Protocol 1:</b> Heat Shock Transformation of <i>E. coli</i></h2><br />
<p>This protocol can be used to transform chemically competent (i.e. from CaCl2) with a miniprepped plasmid or a ligation product.</p><br />
<h5>Note: Never vortex competent cells. Mix cells by gentle shaking.</h5><br />
<ol><br />
<li>Thaw competent cells on ice. These can be prepared using the CaCl2 protocol.</li><br />
<li>Place 20 ul of cells in a pre-chilled Eppendorf tube.<br />
<ul><br />
<li><u>For an Intact Vector:</u> Add 0.5 ul or less to the chilled cells</li><br />
<li><u>For a Ligation Product:</u> Add 2-3 ul to the chilled cells.</li><br />
</ul><br />
</li><br />
<li> Mix gently by flicking the tube.</li><br />
<li> Chill on ice for 10 minutes. <em>This step is optional, but can improve yields when transforming a ligation product.</em></li><br />
<li>Heat shock at 42 &deg;C for 30 seconds.</li><br />
<li>Return to ice for 2 minutes.</li><br />
<li>Add 200 ul LB medium and recover the cells by shaking at 37 &deg;C.<br /><br />
Another rich medium can substitute for the recovery.<br /><br />
The recovery time varies with the antibiotic selection.<br /><br />
Ampicillin: 15-30 minutes<br /><br />
Kanamycin or Spectinomycin: 30-60 minutes<br /><br />
Chloramphenicol: 60-120 minutes <br />
</li><br />
<li>Plate out the cells on selective LB.<br /><br />
Use glass beads to spread the cells.<br /><br />
The volume of cells plated depends on what is being transformed.<br /><br />
<ul><br />
<li><u>For an Intact Vector:</u> High transformation efficiencies are expected. Plating out 10 ul of recovered cells should produce many colonies.</li><br />
<li><u>For a Ligation Product:</u> Lower transformation efficiencies are expected. Therefore you can plate the entire 200 ul volume of recovered cells.</li><br />
</ul><br />
Note: 200 ul is the maximum volume of liquid that an LB plate can absorb.<br />
</li><br />
<li>Incubate at 37 &deg;C. Transformants should appear within 12 hrs.</li><br />
</ol><br />
<br /><br />
<br /><br />
<br />
<h2> <b> Protocol 2:</b> CaCl2 Competent Cells </h2><br />
<br />
<p>This protocol makes 4 ml of competent cells, and can be easily scaled up to make more. The cells are typically stored in 110 ul aliquots, so this will make about 35 tubes. A typical transformation uses 20 ul of cells.<br />
<br />
<h5> Note: Never vortex competent cells. Resuspend by pipetting with large Pasteur pipettes.</h5><br />
<br />
<ol><br />
<p><b><u>The night before:</u></b></p><br />
<li>The night before, inoculate a 5 ml culture and grow overnight with selection.<br />
<p><b><u>The day of:</u></b></p><br />
<li> Dilute cells ~ 1:200 into selective media.<br>For this example add 250 ul to 50 ml of selective media.<br>Note: The protocol is easily scaled to increase the number of cells.<br />
<li> Grow the cells to an OD600 of 0.6 – 0.7.<br />
<br>Use a large flask, 500ml, for good aeration.<br />
<br>Use a baffled flask for fastest growth.<br />
<br>This takes about 3 hours depending on the cells.<br />
<br>Medium-heavy cloudiness by eye is fine.<br />
<li>Spin down the cells at 4 ºC, 4000 rpm, 15 minutes.<br />
Note: Keep the cells at 4 ºC from now on.<br />
<li>Resuspend cells in 15 ml, ice-cold 100 mM CaCl2. <br />
Leave on ice 4 hours to overnight.<br />
<li>Spin down the cells at 4 ºC, 4000 rpm, 15 minutes.<br />
<li>Resuspend cells in 4 ml, ice-cold 100 mM CaCl2 + 15% glycerol.<br />
<li>Aliquot into pre-chilled Eppendorf tubes. Use immediately or store at -80ºC.<br /><br />
Note: Frozen cells are only good once.Do not refreeze cells once thawed.<br />
</ol><br />
<br /><br />
<br /><br />
</body><br />
<br />
<h2> <b> Protocol 3:</b> Glycerol Stocks </h2><br />
<br />
<ol><br />
<br />
<li>Pick Single colonies from agar plates<br />
<li>Innoculate 5ml LB broth overnight.<br />
<li>Add 750ml of overnight culture to 250ml of 60% glycerol in a cryotube.<br />
<li>Make two sets of Glycerol stocks freeze one at -20ºC and the other at -80ºC.<br />
</ol><br />
<br /><br />
<br /><br />
<br />
<h2> <b> Protocol 4: </b> Electroporation </h2><br />
<br />
<ol><br />
<b>Preparation of Electrocompetent Cells</b><br /><br />
Note: Competent cells should never be vortexted, as this will cause them to lyse <br /><br />
and release salts into the media. Resuspend cells by pipeting up and down with a large <br /><br />
pasteur pipet. Once they are chilled, cells should be continuously cold.<br /><br />
<br /><br />
<li>The night before the transformation, start an overnight culture of cells.<br /><br />
5 ml LB Amp.<br /><br />
<br /><br />
<li>The day of the transformation, dilute the cells 100X.<br /><br />
100 ml LB Amp.<br /><br />
Grow at 30&deg;C for about 90 minutes.<br /><br />
<br /><br />
<li>Harvest the cells.<br /><br />
When the cells reach an OD600 of between 0.6 and 0.8.<br /><br />
Split the culture into 2x 50 ml falcon tubes, on ice.<br /><br />
Centrifuge at 4 &deg;C for 10 min at 4000 rpm.<br /><br />
<br /><br />
<li>Wash and combine the cells.<br /><br />
Remove the supernatant.<br /><br />
Resuspend the cells in 2x 25 ml of ice cold water.<br /><br />
Combine the volumes in a single 50 ml falcon tube.<br /><br />
<br /><br />
<li>Wash the cells 2 more times.<br /><br />
Centrifuge at 4 &deg;C for 10 min at 4000 rpm.<br /><br />
Resuspend in 50 ml of ice cold water.<br /><br />
Repeat.<br /><br />
<br /><br />
<li>Wash and concentrate the cells for electroporation.<br /><br />
Centrifuge at 4 &deg;C for 10 min at 4000 rpm.<br /><br />
Resuspend in 1-2 ml of ice cold water.<br /><br />
We will use 200 ul of washed cells per transformation.<br /><br />
</ol><br />
<ol><br />
<br /><br />
<b>Dialysis of PCR or Digestion Products</b><br /><br />
Note: DNA for electroporation must be free of salts to avoid arcing.<br /><br />
<br /><br />
<li>Float a filter in a Petri dish filled with water.<br /><br />
Millipore membrane filter 0.025 uM.<br /><br />
<br /><br />
<li>Pipet one drop of PCR product onto the filter.<br /><br />
200 ng is needed per transformation.<br /><br />
20 - 100 ul fits well on one filter.<br /><br />
<br /><br />
<li>Collect the drop after 30 - 45 minutes.<br /><br />
The volume will change, but the DNA is not lost.<br />
</ol><br />
<br /><br />
<br /><br />
<br />
<h2> <b> Protocol 5:</b> Miniprep </h2><br />
<br />
<h3><b>Miniprep using <i>Thermo Scientific GeneJET Plasmid Miniprep Kit</i></b></h3><br />
<br /><br />
All centrifugations should be carried out in a table-top microcentrifuge at sup12000 x g<br /><br />
<br /><br />
<ol><br />
<li>Resuspend the pelleted cells in 250 ul of the Resuspension Solution. Transfer the cell suspension to a microcentrifuge tube. The bacteria should be resuspended completely by vortexing or pipetting up and down un<br />
til no cell clumps remain.<br />
<br /><br />
<b><i>Note</i></b>.<br />
Ensure RNase A has been added to the Resuspension Solution.<br /><br />
<br /><br />
<li>Add 250 ul of the Lysis Solution and mix thoroughly by inverting the tube 4-6 times until the solution becomes viscous and slightly clear.<br />
<br /><br />
<b><i>Note</i></b>. Do not vortex to avoid shearing of chromosomal DNA. Do not incubate for more than 5 min to avoid denaturation of supercoiled plasmid DNA.<br />
<br /><br />
<br /><br />
<li>Add 350 ul of the Neutralization Solution and mix immediately and thoroughly by inverting the tube 4-6 times.<br />
<br /><br />
<b><i>Note</i></b>.<br />
It is important to mix thoroughly and gently after the addition of the Neutralization Solution to avoid localized precipitation of bacterial cell debris. The neutralized bacterial lysate should become cloudy.<br />
<br /><br />
<br /><br />
<li>Centrifuge for 5 min to pellet cell debris and chromosomal DNA. <br />
<br /><br />
<br /><br />
<li>Transfer the supernatant to the supplied GeneJET spin column by decanting or pipetting. Avoid disturbing or transferring the white precipitate. <br />
<br /><br />
<br /><br />
<li>Centrifuge for 1 min. Discard the flow-through and place the column back into the same collection tube.<br />
<br /><br />
<b><i>Note</i></b>.<br />
Do not add bleach to the flow-through.<br />
<br /><br />
<br /><br />
<li>Add 500 ul of the Wash Solution (diluted with ethanol) to the GeneJET spin column. Centrifuge for 30-60 seconds and discard the flow-through. Place the column back into the same collection tube. <br />
<br /><br />
<br /><br />
<li>Repeat the wash procedure (step 7) using 500 ul of the Wash Solution. <br />
<br /><br />
<br /><br />
<li>Discard the flow-through and centrifuge for an additional 1 min to remove residual Wash Solution. This step is essential to avoid residual ethanol in plasmid preps. <br />
<br /><br />
<br /><br />
<li>Transfer the GeneJETspin column into a fresh 1.5 ml microcentrifuge tube. Add 50 ul of the Elution Bufferto the center of GeneJET spin column membrane to elute the plasmid DNA. Take care not to contact the membrane with the pipette tip. Incubate for 2 min at room tempera ture and centrifuge for 2 min.<br />
<br /><br />
<b><i>Note</i></b>.<br />
An additional elution step (<i>optional</i>) with Elution Buffer or water will recover residual DNA from the membrane and increase the overall yield by 10-20%. For elution of plasmids or cosmids sup20 kb, prewarm Elution Buffer to 70&deg;C before applying to silica membrane.<br />
<br /><br />
<br /><br />
<li>Discard the column and store the purified plasmid DNA at -20&deg;C. <br />
</ol><br />
<br /><br />
<br /><br />
<br />
<h2> <b> Protocol 6:</b> PCR Purification </h2><br />
<br />
<h3><b>PCR purification using <i>Thermo Scientific GeneJET PCR Purification Kit</i></b></h3><br />
<br /><br />
All centrifugations should be carried out in a table-top microcentrifuge at sup12000 x g<br /><br />
<br /><br />
<ol><br />
<li>Add a 1:1 volume of Binding Buffer to completed PCR mixture (e.g. for every 100 uL of reaction mixture, add 100 uL of Binding Buffer). Mix thoroughly. Check the color of the solution. A yellow color indicates an optimal pH for DNA binding. If the color of the solution is orange or violet, add 10 uL of 3 M sodium acetate, pH 5.2 solution and mix. The color of the mix will become yellow.<br />
<br /><br />
<br /><br />
<li><i>Optional</i>: if the DNA fragment is inf 500 bp, add a 1:2 volume of 100% isopropanol(e.g., 100 uL of isopropanol should be added to 100 uL of PCR mixture combined with 100 uL of Binding Buffer). Mix thoroughly.<br /><br />
<b><i>Note</i></b>. If PCR mixture contains primer-dimers, purification without isopropanol is recommended. However, the yield of the target DNA fragment will be lower.<br />
<br /><br />
<br /><br />
<li>Transfer up to 800 uL of the solution from step 1(or optional step 2)to the GeneJET<br />
purification column. Centrifuge for 30-60 s. Discard the flow-through.<br /><br />
<b><i>Note</i></b>. If the total volume exceeds 800 uL, the solution can be added to the column in stages. After the addition of 800 uL of solution, centrifuge the column for 30-60 s and discard flow-through. Repeat until the entire solution has been added to the column membrane.<br />
<br /><br />
<br /><br />
<li>Add 700 uL of Wash Buffer to the GeneJET purification column. Centrifuge for 30-60 s. Discard the flow-through and place the purification column back into the collection tube.<br />
<br /><br />
<br /><br />
<li>Centrifuge the empty GeneJET purification column for an additional 1 min to completely remove any residual wash buffer.<br /><br />
<b><i>Note</i></b>.This step is essential as the presence of residual ethanol in the DNA sample may inhibit subsequent reactions<br />
<br /><br />
<br /><br />
<li>Transfer the GeneJET purification column to a clean 1.5 mL microcentrifuge tube (not included).Add 50 uL of Elution Buffer to the center of the GeneJET purification column membrane and centrifuge for 1 min.<br /><br />
<b><i>Note</i></b>. For low DNA amounts the elution volumes can be reduced to increase DNA concentration. An elution volume between 20-50 uL does not significantly reduce the DNA yield. However, elution volumes less than 10 uL are not recommended. If DNA fragment is inf 10 kb, prewarm Elution Buffer to 65&deg;C before applying to column. If the elution volume is 10 uL and DNA amount is inf 5 ug, incubate column for 1 min at room temperature before centrifugation.<br />
<br /><br />
<br /><br />
<li>Discard the GeneJET purification column and store the purified DNA at -20&deg;C. <br />
</ol><br />
<br /><br />
<br /><br />
<br />
<h2> <b> Protocol 7:</b> Gel Purification </h2><br />
<br />
<h3><b>Gel purification using <i>Thermo Scientific GeneJET Gel Extraction Kit</i></b></h3><br />
<br /><br />
All centrifugations should be carried out in a table-top microcentrifuge at sup12000 x g<br /><br />
<br /><br />
<ol><br />
<li>Excise gel slice containing the DNA fragment using a clean scalpel or razor blade. Cut as close to the DNA as possible to minimize the gel volume. Place the gel slice into a pre-weighed 1.5 ml tube and weigh. Record the weight of the gel slice.<br />
<br /><br />
<b><i>Note</i></b>.<br />
If the purified fragment will be used for cloning reactions, avoid damaging the DNA through UV light exposure. Minimize UV exposure to a few seconds or keep the gel slice on a glass or plastic plate during UV illumination.<br />
<br /><br />
<br /><br />
<li>Add 1:1 volume of Binding Buffer to the gel slice (volume: weight)(e.g., add 100 ul of Binding Buffer for every 100 mg of agarose gel).<br />
<br /><br />
<b><i>Note</i></b>.<br />
For gels with an agarose content greater than 2%, a dd 2:1 volumes of Binding Buffer to the gel slice.<br />
<br /><br />
<br /><br />
<li>Incubate the gel mixture at 50-60&deg;C for 10 min or until the gel slice is completely dissolved. Mix the tube by inversion every few minutes to facilitate the melting process. Ensure that the gel is completely dissolved. Vortex the gel mixture briefly before loading on the column. Check the color of the solution. A yellow color indicates an optimal pH for DNA binding. If the color of the solution is orange or violet, add 10 ul of 3 M sodium acetate, pH 5.2 solution and mix. The color of the mix will become yellow.<br />
<br /><br />
<br /><br />
<li><i>Optional</i>: use this step only when DNA fragment is inf 500 bp or sup10 kb long. If the DNA fragment is inf 500 bp, add a 1:2 volume of 100% isopropanol to the so lubilized gel solution (e.g. 100 ul of isopropanol should be added to 100 mg gel slice solubilized in 100 ul of Binding Buffer). Mix thoroughly. If the DNA fragment is sup10 kb , add a 1:2 volume of water to the solubilized gel solution (e.g. 100 ul of water should be added to 100 mg gel slice solubilized in 100 ul of Binding Buffer). Mix thoroughly. <br />
<br /><br />
<br /><br />
<li>Transfer up to 800 ul of the solubilized gel solution (from step 3 or 4) to the GeneJET purification column. Centrifuge for 1 min. Discard the flow-through and place the column back into the same collection tube.<br />
<br /><br />
<b><i>Note</i></b>. If the total volume exceeds 800 ul, the solution can be added to the column in stages. After each application, centrifuge the column for 30-60 s and discard the flow-through aftereach spin. Repeat until the entire volume has been applied to the column membrane. Do not exceed 1 g of total agarose gel per column.<br />
<br /><br />
<br /><br />
<li><i>Optional</i>: use this additional binding step only if the purified DNA will be used for sequencing. Add 100 ul of Binding Buffer to the GeneJET purification column. Centrifuge for 1 min. Discard the flow-through and place the column back into the same collection tube.<br />
<br /><br />
<br /><br />
<li>Centrifuge the empty GeneJET purification column for an additional 1 min to completely remove residual wash buffer.<br />
<br /><br />
<b><i>Note</i></b>. This step is essential to avoid residual ethanol in the purified DNA solution. The presence of ethanol in the DNA sample may inhibit downstream enzymatic reactions.<br />
<br /><br />
<br /><br />
<li>Transfer the GeneJET purification column into a clean 1.5 ml microcentrifuge tube (not included). Add 50 ul of Elution Buffer to the center of the purification column membrane. Centrifuge for 1 min.<br />
<br /><br />
<b><i>Note</i></b>. For low DNA amounts the elution volumes can be reduced to increase DNA concentration. An elution volume between 20-50 ul does not significantly reduce the DNA yield. However, elution volumes less than 10 ul are not recommended. If DNA fragment is sup10 kb, prewarm Elution Buffer to 65&deg;C before applying to column. If the elution volume is 10 ul and DNA amount is inf5 ug, incubate column for 1 min at room temperature before centrifugation.<br />
<br /><br />
<br /><br />
<li>Discard the GeneJET purification column and store the purified DNA at -20&deg;C.<br />
</ol><br />
<br /><br />
<br /><br />
<br />
<div><br />
<h2> <b> Protocol 8: P1 Transduction</b></h2><br />
</div><br />
<div><br />
<br/><br />
</div><br />
<div><br />
This protocol uses a phage to transfer a marker from a donor strain to a recipient strain. The phage head packages about 90 kb of DNA, so donor DNA near<br />
the marker is also transferred. Note that this can cause problems if you are working with several markers that are very close together.<br />
</div><br />
<div><br />
<br/><br />
</div><br />
<div><br />
This protocol uses a phage to transfer a marker from a donor strain to a recipient strain. The phage head packages about 90 kb of DNA, so donor DNA near<br />
the marker is also transferred. Note that this can cause problems if you are working with several markers that are very close together.<br />
</div><br />
<div><br />
<br/><br />
</div><br />
<div><br />
<strong><u>Lysate preparation</u></strong><br />
</div><br />
<div><br />
<br/><br />
</div><br />
<div><br />
<strong>Note: P1 phage should be stored at 4 C. It can't be frozen.</strong><br />
</div><br />
<div><br />
<br/><br />
</div><br />
<div><br />
1) The night before, start a 5 mL culture of the donor strain in selective LB.<br />
</div><br />
<div><br />
<br/><br />
</div><br />
<div><br />
2) The day of, dilute the donor strain 1:100 into Phage Lysis medium.<br />
</div><br />
<div><br />
50 ul of cells in 5 mL LB<br />
<br/><br />
</div><br />
<div><br />
+ 50 uL of 20% Glucose<br />
<br/><br />
</div><br />
<div><br />
+ 50 uL of 1M MgSO4<br />
<br/><br />
</div><br />
<div><br />
+ 25 ul of 1M CaCl2<br />
<br/><br />
</div><br />
<div><br />
No antibiotics<br />
<br/><br />
</div><br />
<div><br />
<br/><br />
</div><br />
<div><br />
3) Incubate at 37 C for 1 hour.<br />
<br/><br />
</div><br />
<div><br />
<br/><br />
</div><br />
<div><br />
4) Add 50 ul of P1 phage lysate.<br />
<br/><br />
</div><br />
<div><br />
Monitor the culture for 1-3 hours.<br />
<br/><br />
</div><br />
<div><br />
The culture should become cloudy, then clear following lysis.<br />
<br/><br />
</div><br />
<div><br />
<br/><br />
</div><br />
<div><br />
5) Add 500 ul of chloroform to the lysate and vortex.<br />
<br/><br />
</div><br />
<div><br />
<br/><br />
</div><br />
<div><br />
6) Centrifuge at max speed for 1 minute to clear the cell debris.<br />
<br/><br />
</div><br />
<div><br />
Collect the supernatant.<br />
</div><br />
<div><br />
<br/><br />
</div><br />
<div><br />
7) Phage lysate can be stored indefinitely at 4 C. Freezing will destroy the phage.<br />
</div><br />
<div><br />
</div><br />
<div><br />
<strong><u>Transduction</u></strong><br />
</div><br />
<div><br />
<br/><br />
</div><br />
<div><br />
1) The night before, start a 5 ml culture of the recipient strain in selective LB.<br />
</div><br />
<div><br />
<br/><br />
</div><br />
<div><br />
2) The day of, harvest the cells by centrifugation.<br />
<br/><br />
</div><br />
<div><br />
6000 rpm for 2 min.<br />
<br/><br />
</div><br />
<div><br />
<br/><br />
</div><br />
<div><br />
3) Resuspend in original culture volume in 5 mL Phage Infection LB.<br />
<br/><br />
</div><br />
<div><br />
5 mL LB<br />
<br/><br />
</div><br />
<div><br />
+ 50 uL of 1M MgSO4<br />
<br/><br />
</div><br />
<div><br />
+ 25 ul of 1M CaCl2<br />
<br/><br />
</div><br />
<div><br />
<br/><br />
</div><br />
<div><br />
4) Transfer 100 uL of donor P1 lysate per transformation to a 1.5 mL tube.<br />
<br/><br />
</div><br />
<div><br />
Incubate at 37 C for 30 minutes.<br />
<br/><br />
</div><br />
<div><br />
This allows the residual chloroform to evaporate.<br />
<br/><br />
</div><br />
<div><br />
<br/><br />
</div><br />
<div><br />
5) Set up 4 reactions for each transduction<br />
<br/><br />
</div><br />
<div><br />
1) 100 uL Donor Lysate 2) 10 uL Donor Lysate 3) 100 uL Donor Lysate 4) 100 uL Plain Lb<br />
<br/><br />
</div><br />
<div><br />
100 uL Recipient Cells 190 uL Recipient Cells 100 uL Plain LB 100 uL Recipient Cells<br />
</div><br />
<div><br />
<br/><br />
</div><br />
<div><br />
6) Incubate at 37 C for 30 minutes.<br />
<br/><br />
</div><br />
<div><br />
<br/><br />
</div><br />
<div><br />
7) Stop the infection with 200 uL of 1 M Sodium Citrate (pH 5.5).<br />
<br/><br />
</div><br />
<div><br />
<br/><br />
</div><br />
<div><br />
8) Add 1 mL LB and recover the cells for 1-2 hours.<br />
<br/><br />
</div><br />
<div><br />
<br/><br />
</div><br />
<div><br />
9) Spin the cells down and resuspend for plating.<br />
<br/><br />
</div><br />
<div><br />
100 ul LB + 10 uL of 1 M Sodium Citrate (pH 5.5)<br />
<br/><br />
</div><br />
<div><br />
10) Plate on selective LB.<br />
</div><br />
</ol><br />
<br /><br />
<br /><br />
<br />
</div><br />
</html><br />
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<h2> <b> Protocol 1:</b> Heat Shock Transformation of <i>E. coli</i></h2><br />
<p>This protocol can be used to transform chemically competent (i.e. from CaCl2) with a miniprepped plasmid or a ligation product.</p><br />
<h5>Note: Never vortex competent cells. Mix cells by gentle shaking.</h5><br />
<ol><br />
<li>Thaw competent cells on ice. These can be prepared using the CaCl2 protocol.</li><br />
<li>Place 20 ul of cells in a pre-chilled Eppendorf tube.<br />
<ul><br />
<li><u>For an Intact Vector:</u> Add 0.5 ul or less to the chilled cells</li><br />
<li><u>For a Ligation Product:</u> Add 2-3 ul to the chilled cells.</li><br />
</ul><br />
</li><br />
<li> Mix gently by flicking the tube.</li><br />
<li> Chill on ice for 10 minutes. <em>This step is optional, but can improve yields when transforming a ligation product.</em></li><br />
<li>Heat shock at 42 &deg;C for 30 seconds.</li><br />
<li>Return to ice for 2 minutes.</li><br />
<li>Add 200 ul LB medium and recover the cells by shaking at 37 &deg;C.<br /><br />
Another rich medium can substitute for the recovery.<br /><br />
The recovery time varies with the antibiotic selection.<br /><br />
Ampicillin: 15-30 minutes<br /><br />
Kanamycin or Spectinomycin: 30-60 minutes<br /><br />
Chloramphenicol: 60-120 minutes <br />
</li><br />
<li>Plate out the cells on selective LB.<br /><br />
Use glass beads to spread the cells.<br /><br />
The volume of cells plated depends on what is being transformed.<br /><br />
<ul><br />
<li><u>For an Intact Vector:</u> High transformation efficiencies are expected. Plating out 10 ul of recovered cells should produce many colonies.</li><br />
<li><u>For a Ligation Product:</u> Lower transformation efficiencies are expected. Therefore you can plate the entire 200 ul volume of recovered cells.</li><br />
</ul><br />
Note: 200 ul is the maximum volume of liquid that an LB plate can absorb.<br />
</li><br />
<li>Incubate at 37 &deg;C. Transformants should appear within 12 hrs.</li><br />
</ol><br />
<br /><br />
<br /><br />
<br />
<h2> <b> Protocol 2:</b> CaCl2 Competent Cells </h2><br />
<br />
<p>This protocol makes 4 ml of competent cells, and can be easily scaled up to make more. The cells are typically stored in 110 ul aliquots, so this will make about 35 tubes. A typical transformation uses 20 ul of cells.<br />
<br />
<h5> Note: Never vortex competent cells. Resuspend by pipetting with large Pasteur pipettes.</h5><br />
<br />
<ol><br />
<p><b><u>The night before:</u></b></p><br />
<li>The night before, inoculate a 5 ml culture and grow overnight with selection.<br />
<p><b><u>The day of:</u></b></p><br />
<li> Dilute cells ~ 1:200 into selective media.<br>For this example add 250 ul to 50 ml of selective media.<br>Note: The protocol is easily scaled to increase the number of cells.<br />
<li> Grow the cells to an OD600 of 0.6 – 0.7.<br />
<br>Use a large flask, 500ml, for good aeration.<br />
<br>Use a baffled flask for fastest growth.<br />
<br>This takes about 3 hours depending on the cells.<br />
<br>Medium-heavy cloudiness by eye is fine.<br />
<li>Spin down the cells at 4 ºC, 4000 rpm, 15 minutes.<br />
Note: Keep the cells at 4 ºC from now on.<br />
<li>Resuspend cells in 15 ml, ice-cold 100 mM CaCl2. <br />
Leave on ice 4 hours to overnight.<br />
<li>Spin down the cells at 4 ºC, 4000 rpm, 15 minutes.<br />
<li>Resuspend cells in 4 ml, ice-cold 100 mM CaCl2 + 15% glycerol.<br />
<li>Aliquot into pre-chilled Eppendorf tubes. Use immediately or store at -80ºC.<br /><br />
Note: Frozen cells are only good once.Do not refreeze cells once thawed.<br />
</ol><br />
<br /><br />
<br /><br />
</body><br />
<br />
<h2> <b> Protocol 3:</b> Glycerol Stocks </h2><br />
<br />
<ol><br />
<br />
<li>Pick Single colonies from agar plates<br />
<li>Innoculate 5ml LB broth overnight.<br />
<li>Add 750ml of overnight culture to 250ml of 60% glycerol in a cryotube.<br />
<li>Make two sets of Glycerol stocks freeze one at -20ºC and the other at -80ºC.<br />
</ol><br />
<br /><br />
<br /><br />
<br />
<h2> <b> Protocol 4: </b> Electroporation </h2><br />
<br />
<ol><br />
<b>Preparation of Electrocompetent Cells</b><br /><br />
Note: Competent cells should never be vortexted, as this will cause them to lyse <br /><br />
and release salts into the media. Resuspend cells by pipeting up and down with a large <br /><br />
pasteur pipet. Once they are chilled, cells should be continuously cold.<br /><br />
<br /><br />
<li>The night before the transformation, start an overnight culture of cells.<br /><br />
5 ml LB Amp.<br /><br />
<br /><br />
<li>The day of the transformation, dilute the cells 100X.<br /><br />
100 ml LB Amp.<br /><br />
Grow at 30&deg;C for about 90 minutes.<br /><br />
<br /><br />
<li>Harvest the cells.<br /><br />
When the cells reach an OD600 of between 0.6 and 0.8.<br /><br />
Split the culture into 2x 50 ml falcon tubes, on ice.<br /><br />
Centrifuge at 4 &deg;C for 10 min at 4000 rpm.<br /><br />
<br /><br />
<li>Wash and combine the cells.<br /><br />
Remove the supernatant.<br /><br />
Resuspend the cells in 2x 25 ml of ice cold water.<br /><br />
Combine the volumes in a single 50 ml falcon tube.<br /><br />
<br /><br />
<li>Wash the cells 2 more times.<br /><br />
Centrifuge at 4 &deg;C for 10 min at 4000 rpm.<br /><br />
Resuspend in 50 ml of ice cold water.<br /><br />
Repeat.<br /><br />
<br /><br />
<li>Wash and concentrate the cells for electroporation.<br /><br />
Centrifuge at 4 &deg;C for 10 min at 4000 rpm.<br /><br />
Resuspend in 1-2 ml of ice cold water.<br /><br />
We will use 200 ul of washed cells per transformation.<br /><br />
</ol><br />
<ol><br />
<br /><br />
<b>Dialysis of PCR or Digestion Products</b><br /><br />
Note: DNA for electroporation must be free of salts to avoid arcing.<br /><br />
<br /><br />
<li>Float a filter in a Petri dish filled with water.<br /><br />
Millipore membrane filter 0.025 uM.<br /><br />
<br /><br />
<li>Pipet one drop of PCR product onto the filter.<br /><br />
200 ng is needed per transformation.<br /><br />
20 - 100 ul fits well on one filter.<br /><br />
<br /><br />
<li>Collect the drop after 30 - 45 minutes.<br /><br />
The volume will change, but the DNA is not lost.<br />
</ol><br />
<br /><br />
<br /><br />
<br />
<h2> <b> Protocol 5:</b> Miniprep </h2><br />
<br />
<h3><b>Miniprep using <i>Thermo Scientific GeneJET Plasmid Miniprep Kit</i></b></h3><br />
<br /><br />
All centrifugations should be carried out in a table-top microcentrifuge at sup12000 x g<br /><br />
<br /><br />
<ol><br />
<li>Resuspend the pelleted cells in 250 ul of the Resuspension Solution. Transfer the cell suspension to a microcentrifuge tube. The bacteria should be resuspended completely by vortexing or pipetting up and down un<br />
til no cell clumps remain.<br />
<br /><br />
<b><i>Note</i></b>.<br />
Ensure RNase A has been added to the Resuspension Solution.<br /><br />
<br /><br />
<li>Add 250 ul of the Lysis Solution and mix thoroughly by inverting the tube 4-6 times until the solution becomes viscous and slightly clear.<br />
<br /><br />
<b><i>Note</i></b>. Do not vortex to avoid shearing of chromosomal DNA. Do not incubate for more than 5 min to avoid denaturation of supercoiled plasmid DNA.<br />
<br /><br />
<br /><br />
<li>Add 350 ul of the Neutralization Solution and mix immediately and thoroughly by inverting the tube 4-6 times.<br />
<br /><br />
<b><i>Note</i></b>.<br />
It is important to mix thoroughly and gently after the addition of the Neutralization Solution to avoid localized precipitation of bacterial cell debris. The neutralized bacterial lysate should become cloudy.<br />
<br /><br />
<br /><br />
<li>Centrifuge for 5 min to pellet cell debris and chromosomal DNA. <br />
<br /><br />
<br /><br />
<li>Transfer the supernatant to the supplied GeneJET spin column by decanting or pipetting. Avoid disturbing or transferring the white precipitate. <br />
<br /><br />
<br /><br />
<li>Centrifuge for 1 min. Discard the flow-through and place the column back into the same collection tube.<br />
<br /><br />
<b><i>Note</i></b>.<br />
Do not add bleach to the flow-through.<br />
<br /><br />
<br /><br />
<li>Add 500 ul of the Wash Solution (diluted with ethanol) to the GeneJET spin column. Centrifuge for 30-60 seconds and discard the flow-through. Place the column back into the same collection tube. <br />
<br /><br />
<br /><br />
<li>Repeat the wash procedure (step 7) using 500 ul of the Wash Solution. <br />
<br /><br />
<br /><br />
<li>Discard the flow-through and centrifuge for an additional 1 min to remove residual Wash Solution. This step is essential to avoid residual ethanol in plasmid preps. <br />
<br /><br />
<br /><br />
<li>Transfer the GeneJETspin column into a fresh 1.5 ml microcentrifuge tube. Add 50 ul of the Elution Bufferto the center of GeneJET spin column membrane to elute the plasmid DNA. Take care not to contact the membrane with the pipette tip. Incubate for 2 min at room tempera ture and centrifuge for 2 min.<br />
<br /><br />
<b><i>Note</i></b>.<br />
An additional elution step (<i>optional</i>) with Elution Buffer or water will recover residual DNA from the membrane and increase the overall yield by 10-20%. For elution of plasmids or cosmids sup20 kb, prewarm Elution Buffer to 70&deg;C before applying to silica membrane.<br />
<br /><br />
<br /><br />
<li>Discard the column and store the purified plasmid DNA at -20&deg;C. <br />
</ol><br />
<br /><br />
<br /><br />
<br />
<h2> <b> Protocol 6:</b> PCR Purification </h2><br />
<br />
<h3><b>PCR purification using <i>Thermo Scientific GeneJET PCR Purification Kit</i></b></h3><br />
<br /><br />
All centrifugations should be carried out in a table-top microcentrifuge at sup12000 x g<br /><br />
<br /><br />
<ol><br />
<li>Add a 1:1 volume of Binding Buffer to completed PCR mixture (e.g. for every 100 uL of reaction mixture, add 100 uL of Binding Buffer). Mix thoroughly. Check the color of the solution. A yellow color indicates an optimal pH for DNA binding. If the color of the solution is orange or violet, add 10 uL of 3 M sodium acetate, pH 5.2 solution and mix. The color of the mix will become yellow.<br />
<br /><br />
<br /><br />
<li><i>Optional</i>: if the DNA fragment is inf 500 bp, add a 1:2 volume of 100% isopropanol(e.g., 100 uL of isopropanol should be added to 100 uL of PCR mixture combined with 100 uL of Binding Buffer). Mix thoroughly.<br /><br />
<b><i>Note</i></b>. If PCR mixture contains primer-dimers, purification without isopropanol is recommended. However, the yield of the target DNA fragment will be lower.<br />
<br /><br />
<br /><br />
<li>Transfer up to 800 uL of the solution from step 1(or optional step 2)to the GeneJET<br />
purification column. Centrifuge for 30-60 s. Discard the flow-through.<br /><br />
<b><i>Note</i></b>. If the total volume exceeds 800 uL, the solution can be added to the column in stages. After the addition of 800 uL of solution, centrifuge the column for 30-60 s and discard flow-through. Repeat until the entire solution has been added to the column membrane.<br />
<br /><br />
<br /><br />
<li>Add 700 uL of Wash Buffer to the GeneJET purification column. Centrifuge for 30-60 s. Discard the flow-through and place the purification column back into the collection tube.<br />
<br /><br />
<br /><br />
<li>Centrifuge the empty GeneJET purification column for an additional 1 min to completely remove any residual wash buffer.<br /><br />
<b><i>Note</i></b>.This step is essential as the presence of residual ethanol in the DNA sample may inhibit subsequent reactions<br />
<br /><br />
<br /><br />
<li>Transfer the GeneJET purification column to a clean 1.5 mL microcentrifuge tube (not included).Add 50 uL of Elution Buffer to the center of the GeneJET purification column membrane and centrifuge for 1 min.<br /><br />
<b><i>Note</i></b>. For low DNA amounts the elution volumes can be reduced to increase DNA concentration. An elution volume between 20-50 uL does not significantly reduce the DNA yield. However, elution volumes less than 10 uL are not recommended. If DNA fragment is inf 10 kb, prewarm Elution Buffer to 65&deg;C before applying to column. If the elution volume is 10 uL and DNA amount is inf 5 ug, incubate column for 1 min at room temperature before centrifugation.<br />
<br /><br />
<br /><br />
<li>Discard the GeneJET purification column and store the purified DNA at -20&deg;C. <br />
</ol><br />
<br /><br />
<br /><br />
<br />
<h2> <b> Protocol 7:</b> Gel Purification </h2><br />
<br />
<h3><b>Gel purification using <i>Thermo Scientific GeneJET Gel Extraction Kit</i></b></h3><br />
<br /><br />
All centrifugations should be carried out in a table-top microcentrifuge at sup12000 x g<br /><br />
<br /><br />
<ol><br />
<li>Excise gel slice containing the DNA fragment using a clean scalpel or razor blade. Cut as close to the DNA as possible to minimize the gel volume. Place the gel slice into a pre-weighed 1.5 ml tube and weigh. Record the weight of the gel slice.<br />
<br /><br />
<b><i>Note</i></b>.<br />
If the purified fragment will be used for cloning reactions, avoid damaging the DNA through UV light exposure. Minimize UV exposure to a few seconds or keep the gel slice on a glass or plastic plate during UV illumination.<br />
<br /><br />
<br /><br />
<li>Add 1:1 volume of Binding Buffer to the gel slice (volume: weight)(e.g., add 100 ul of Binding Buffer for every 100 mg of agarose gel).<br />
<br /><br />
<b><i>Note</i></b>.<br />
For gels with an agarose content greater than 2%, a dd 2:1 volumes of Binding Buffer to the gel slice.<br />
<br /><br />
<br /><br />
<li>Incubate the gel mixture at 50-60&deg;C for 10 min or until the gel slice is completely dissolved. Mix the tube by inversion every few minutes to facilitate the melting process. Ensure that the gel is completely dissolved. Vortex the gel mixture briefly before loading on the column. Check the color of the solution. A yellow color indicates an optimal pH for DNA binding. If the color of the solution is orange or violet, add 10 ul of 3 M sodium acetate, pH 5.2 solution and mix. The color of the mix will become yellow.<br />
<br /><br />
<br /><br />
<li><i>Optional</i>: use this step only when DNA fragment is inf 500 bp or sup10 kb long. If the DNA fragment is inf 500 bp, add a 1:2 volume of 100% isopropanol to the so lubilized gel solution (e.g. 100 ul of isopropanol should be added to 100 mg gel slice solubilized in 100 ul of Binding Buffer). Mix thoroughly. If the DNA fragment is sup10 kb , add a 1:2 volume of water to the solubilized gel solution (e.g. 100 ul of water should be added to 100 mg gel slice solubilized in 100 ul of Binding Buffer). Mix thoroughly. <br />
<br /><br />
<br /><br />
<li>Transfer up to 800 ul of the solubilized gel solution (from step 3 or 4) to the GeneJET purification column. Centrifuge for 1 min. Discard the flow-through and place the column back into the same collection tube.<br />
<br /><br />
<b><i>Note</i></b>. If the total volume exceeds 800 ul, the solution can be added to the column in stages. After each application, centrifuge the column for 30-60 s and discard the flow-through aftereach spin. Repeat until the entire volume has been applied to the column membrane. Do not exceed 1 g of total agarose gel per column.<br />
<br /><br />
<br /><br />
<li><i>Optional</i>: use this additional binding step only if the purified DNA will be used for sequencing. Add 100 ul of Binding Buffer to the GeneJET purification column. Centrifuge for 1 min. Discard the flow-through and place the column back into the same collection tube.<br />
<br /><br />
<br /><br />
<li>Centrifuge the empty GeneJET purification column for an additional 1 min to completely remove residual wash buffer.<br />
<br /><br />
<b><i>Note</i></b>. This step is essential to avoid residual ethanol in the purified DNA solution. The presence of ethanol in the DNA sample may inhibit downstream enzymatic reactions.<br />
<br /><br />
<br /><br />
<li>Transfer the GeneJET purification column into a clean 1.5 ml microcentrifuge tube (not included). Add 50 ul of Elution Buffer to the center of the purification column membrane. Centrifuge for 1 min.<br />
<br /><br />
<b><i>Note</i></b>. For low DNA amounts the elution volumes can be reduced to increase DNA concentration. An elution volume between 20-50 ul does not significantly reduce the DNA yield. However, elution volumes less than 10 ul are not recommended. If DNA fragment is sup10 kb, prewarm Elution Buffer to 65&deg;C before applying to column. If the elution volume is 10 ul and DNA amount is inf5 ug, incubate column for 1 min at room temperature before centrifugation.<br />
<br /><br />
<br /><br />
<li>Discard the GeneJET purification column and store the purified DNA at -20&deg;C.<br />
</ol><br />
<br /><br />
<br /><br />
<br />
<div><br />
<h2> <b> Protocol 8: P1 Transduction</b></h2><br />
</div><br />
<div><br />
<br/><br />
</div><br />
<div><br />
This protocol uses a phage to transfer a marker from a donor strain to a recipient strain. The phage head packages about 90 kb of DNA, so donor DNA near<br />
the marker is also transferred. Note that this can cause problems if you are working with several markers that are very close together.<br />
</div><br />
<div><br />
<br/><br />
</div><br />
<div><br />
<strong><u>Lysate preparation</u></strong><br />
</div><br />
<div><br />
<br/><br />
</div><br />
<div><br />
<strong>Note: P1 phage should be stored at 4 C. It can't be frozen.</strong><br />
</div><br />
<div><br />
<br/><br />
</div><br />
<div><br />
1) The night before, start a 5 mL culture of the donor strain in selective LB.<br />
</div><br />
<div><br />
2) The day of, dilute the donor strain 1:100 into Phage Lysis medium.<br />
</div><br />
<div><br />
50 ul of cells in 5 mL LB<br />
</div><br />
<div><br />
+ 50 uL of 20% Glucose<br />
<br/><br />
</div><br />
<div><br />
+ 50 uL of 1M MgSO4<br />
<br/><br />
</div><br />
<div><br />
+ 25 ul of 1M CaCl2<br />
<br/><br />
</div><br />
<div><br />
No antibiotics<br />
<br/><br />
</div><br />
<div><br />
3) Incubate at 37 C for 1 hour.<br />
<br/><br />
</div><br />
<div><br />
4) Add 50 ul of P1 phage lysate.<br />
<br/><br />
</div><br />
<div><br />
Monitor the culture for 1-3 hours.<br />
<br/><br />
</div><br />
<div><br />
The culture should become cloudy, then clear following lysis.<br />
<br/><br />
</div><br />
<div><br />
5) Add 500 ul of chloroform to the lysate and vortex.<br />
<br/><br />
</div><br />
<div><br />
6) Centrifuge at max speed for 1 minute to clear the cell debris.<br />
<br/><br />
</div><br />
<div><br />
Collect the supernatant.<br />
</div><br />
<div><br />
7) Phage lysate can be stored indefinitely at 4 C. Freezing will destroy the phage.<br />
</div><br />
<div><br />
<br/><br />
</div><br />
<div><br />
<strong><u>Transduction</u></strong><br />
</div><br />
<div><br />
<br/><br />
</div><br />
<div><br />
1) The night before, start a 5 ml culture of the recipient strain in selective LB.<br />
</div><br />
<div><br />
2) The day of, harvest the cells by centrifugation.<br />
<br/><br />
</div><br />
<div><br />
6000 rpm for 2 min.<br />
<br/><br />
</div><br />
<div><br />
3) Resuspend in original culture volume in 5 mL Phage Infection LB.<br />
<br/><br />
</div><br />
<div><br />
5 mL LB<br />
<br/><br />
</div><br />
<div><br />
+ 50 uL of 1M MgSO4<br />
<br/><br />
</div><br />
<div><br />
+ 25 ul of 1M CaCl2<br />
<br/><br />
</div><br />
<div><br />
4) Transfer 100 uL of donor P1 lysate per transformation to a 1.5 mL tube.<br />
<br/><br />
</div><br />
<div><br />
Incubate at 37 C for 30 minutes.<br />
<br/><br />
</div><br />
<div><br />
This allows the residual chloroform to evaporate.<br />
<br/><br />
</div><br />
<div><br />
5) Set up 4 reactions for each transduction<br />
<br/><br />
</div><br />
<div><br />
1) 100 uL Donor Lysate 2) 10 uL Donor Lysate 3) 100 uL Donor Lysate 4) 100 uL Plain Lb<br />
<br/><br />
</div><br />
<div><br />
100 uL Recipient Cells 190 uL Recipient Cells 100 uL Plain LB 100 uL Recipient Cells<br />
</div><br />
<div><br />
<br/><br />
</div><br />
<div><br />
6) Incubate at 37 C for 30 minutes.<br />
</div><br />
<div><br />
7) Stop the infection with 200 uL of 1 M Sodium Citrate (pH 5.5).<br />
<br/><br />
</div><br />
<div><br />
8) Add 1 mL LB and recover the cells for 1-2 hours.<br />
<br/><br />
</div><br />
<div><br />
9) Spin the cells down and resuspend for plating.<br />
<br/><br />
</div><br />
<div><br />
100 ul LB + 10 uL of 1 M Sodium Citrate (pH 5.5)<br />
<br/><br />
</div><br />
<div><br />
10) Plate on selective LB.<br />
</div><br />
</ol><br />
<br /><br />
<br /><br />
<br />
</div><br />
</html><br />
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{{:Team:Paris_Bettencourt/footer}}</div>Idonnyahttp://2013.igem.org/Team:Paris_Bettencourt/BibliographyTeam:Paris Bettencourt/Bibliography2013-10-04T19:21:39Z<p>Idonnya: </p>
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