Team:SydneyUni Australia/Project/Design

From 2013.igem.org

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__NOTOC__
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== '''Gibson'''==
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Gibson Assembly *should* make for a much easier, simpler, rapid assembly of different genes than conventional PCR and cloning, plus there’s much more flexibility for optimisation through gene synthesis. 
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* The assembly works on fragments of DNA with ~30bp of overlapping sequence, which is exposed as 5’ single-stranded overhangs by an exonuclease. A ligase joins the overlapping regions and a polymerase fills in any gaps left by the exonuclease. These enzymes can all work together in a single reaction tube with many different overlapping fragments, making the assembly a very, very simple activity. Gibson Assembly is based on the older technique of [http://nar.oxfordjournals.org/content/32/12/e98.full PCR Assembly], with the similar reliance on the initial construction of 200+bp fragments from smaller oligos, but with a greater degree of sequence fidelity due to less polymerase activity.  
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* Here’s a great introduction from IDT’s magazine, [http://www.idtdna.com/pages/decoded/decoded-articles/core-concepts/decoded/2012/01/10/isothermal-assembly-quick-easy-gene-construction DECODED], and a more in-depth [https://www.idtdna.com/pages/support/technical-vault/webinars/categories/synthetic-genes/rapid-and-reliable-gene-construction webinar].
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* If you’re historically-minded or want more detail, try the [http://diyhpl.us/~bryan/papers2/bio/venter/Enzymatic%20assembly%20of%20DNA%20molecules%20up%20to%20several%20hundred%20kilobases.pdf original paper] in which Gibson Assembly was described - or one of the coolest and most famous applications of Gibson, building a [http://www.ncbi.nlm.nih.gov/books/NBK84435/ synthetic genome].
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== '''Design'''==
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text-weight: bolder;
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text-color: #82CA9C;
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=== '''Choice of genes'''===
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'''Mox, chloroethanol dehydrogenase'''
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*In sketches of our project (February-March), we planned to use Mox as the alcohol dehydrogenase converting chloroethanol to chloroacetaldehyde. After a little more research we discovered that this enzyme requires the co-factor PQQ, and unfortunately for us, this co-factor requires many genes for its synthesis, which would have made our constructs too complex (Khairnar et al, 2003).
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*Mox had seemed like an obvious choice because it would be sourced from Xanthobacter autotrophicus GJ10, the most well-documented DCA-degrader ([http://mic.sgmjournals.org/content/133/1/85.full.pdf Janssen et al, 1987])
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== '''Project Results'''==
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'''aldA, chloroacetaldehyde dehydrogenase, dhlB, haloacid dehalogenase, and dhlA, haloalkane dehalogenase'''
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* We used a sequence for aldA from Xanthobacter autrophicus GJ10, but used sequences for dhlB and dhlA from a different strain, Xanthobacter autrophicus EL4, which was isolated and characterised in the Coleman lab. These two genes had been identified and cloned into pUC19 by Jake Munro, a research assistant in the Coleman lab. When our Gibson Assembly ran into problems, we continued to work with dhlB and dhlA from EL4 in our lab and submitted these two genes as [https://2013.igem.org/Team:SydneyUni_Australia/Project/Parts parts].  
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*These genes are shared by a few different species of bacteria that degrade DCA (Janssen et al, 1994), and most have been conventionally characterised by extraction and heterologous expression of a single gene at a time.  
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  <div class="unlink">ToMO degrades DCA</div>
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  <ul>
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<div class="pictext">
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<div class="pictextl" style="height: 100px;">Early in our project we needed to find a suitable monooxygenase to begin degradation of DCA by one of the two degradation pathways. </div>
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<a href="https://static.igem.org/mediawiki/2013/9/90/DCApathwaysHartman.jpg" rel="ibox" title="DCA Degradation Pathways">
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    <li>Toluene-o-xylene monooxygenase (ToMO) from Pseuodomonas stutzeri OX1 has been shown to oxidise xylenes, toluene, benzene, styrene, napthalene (Bertoni et al, 1996) as well as tetrachloroethene, trichloroethene, dichloroethene and vinyl chloride (Shim et al, 2001). The enzyme was optimised for chlorinated ethene degradation (Varder & Wood, 2005), and gifted to our host lab in the plasmid pBS(Kan)ToMO.</li>
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<ul>
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<li>Bertoni, G., Bolognese, F., Galli, E., & Barbieri, P. (1996). Cloning of the genes for and characterization of the early stages of toluene and o-xylene catabolism in Pseudomonas stutzeri OX1. Applied and environmental microbiology, 62(10), 3704-3711.</li>
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<li>Shim, H., Ryoo, D., Barbieri, P., & Wood, T. (2001). Aerobic degradation of mixtures of tetrachloroethylene, trichloroethylene, dichloroethylenes, and vinyl chloride by toluene-o-xylene monooxygenase of Pseudomonas stutzeri OX1. Applied microbiology and biotechnology, 56(1-2), 265-269.</li>
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<li>Vardar, G., & Wood, T. K. (2005). Protein engineering of toluene-o-xylene monooxygenase from Pseudomonas stutzeri OX1 for enhanced chlorinated ethene degradation and o-xylene oxidation. Applied microbiology and biotechnology, 68(4), 510-517.</li>
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    <li>We showed that ToMO can begin degradation of DCA through an assay for chloride ions (link to protocol) released as DCA is converted to chloroacetaldehyde. To our knowledge this has’t been shown by anyone else before. </li>
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<li>This is pretty cool, but during the middle of the year we decided to try synthesising the whole pathway rather than building it by conventional cloning. The length of the ToMO gene cluster meant it was too expensive for us to continue working with it.</li>
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'''ToMO, toluene-o-xylene monooxygenase'''
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<div class="pictext">
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* Early in our project we showed that [https://2013.igem.org/Team:SydneyUni_Australia/Project/Results ToMO can degrade DCA]. This would not only eliminate the need for something like PQQ-synthase, but also make for a shorter, less complex pathway involving just 3 enzymes (a monooxygenase, dehydrogenase and dehalogenase).
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<div class="pictextl" style="height: 100px; line-height: 100px">E. coli expressing ToMO converts indol to an indo-coloured compound:</div>
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* We initially planned to synthesise ToMO, allowing us to remove forbidden restriction sites in the process (EcoRI and PstI). However, due to the sheer size of the monooxygenase cluster (~5kb) we could not afford to have this gene and all of our others synthesised.
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*The gene we worked with initially has been extensively used for protein engineering by [http://fenske.che.psu.edu/faculty/wood/group/publications/pdf/ToMO%20TCE%20mutagenesis%20Vardar[1].pdf Varder & Wood (2005)].
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<a href="https://static.igem.org/mediawiki/2013/9/97/SydneyUni2013_Results_TomoIndigo.jpg" rel="ibox" title="ToMO Cl- Assay">
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<img src="https://static.igem.org/mediawiki/2013/9/97/SydneyUni2013_Results_TomoIndigo.jpg" height="100">
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'''p450, cytochrome p450 monooxygenase'''
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  <div class="unlink">Gibson Assembly Was Problematic</div>
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<ul>
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<li> <b>Progress</b> </li>
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<ul>
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<li> <b>Transformation</b> </li>
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<li>We spent a week (12/9 to 16/9) optimising the transformation of our <a href="https://2013.igem.org/Team:SydneyUni_Australia/Project/Design">Gibson Assembly reaction product</a>. We initially tried transformation into E. Coli EPI300 and yielded no transformants. We suspected that there may be a metabolic burden or harm to cells carrying our correctly assembled product, due to the strong constitutive expression of our designed promoter, Psyn <b>(link to design of gBlocks, Psyn explanation)</b>. To account for this we tried transforming into E. Coli EPI400, which carries plasmids at low copy-number with an inducible increase in copy-number. We also tried incubating and growing cells at room temperature to lessen their growth rate, so that they might be able to better cope with any possible toxicity of the transformed Gibson Assembly reaction product. Neither of these were successful, however, we were able to screen 87 clones by transforming into a different strain, E. Coli TOP10.</li>
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<li><b> Screening </b></li>
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<ul>
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<li>We screened 87 clones for the presence of dhlB, a gene responsible for the breakdown of chloroacetate in our pathway, by incubating resting cells with chloroacetate and chloride assay (link to protocol). A few clones from each type of pathway looked promising, so we proceeded to extract plasmids from these for further investigation.</li>
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<div class="pictextl">dhlB Screening:<br>We expect a band at ~800bp for an amplification of dhlB, the gene in our pathway responsible for the degradation of chloroacetate. We don’t see it any of the 87 clones, which corresponds to our phenotypic assays of the clones, where few of the clones released many Cl- ions from the chloroacetate substrate. </div>
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<a href="https://static.igem.org/mediawiki/2013/4/44/SydneyUniversity2013_results_PCRscreeningdhlBfail.jpg" rel="ibox" title="dhlB Screening">
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<li>Later on <b>(link to calendar, it was about a week later, after a set of primers had arrived)</b>, we also tried screening for dhlB by PCR. We amplicon of interest would have spanned only a single overlap during Gibson Assembly, yet we failed to find a single clone containing the assembled (or misassembled) gene dhlB.</li>
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<li> <b>Plasmid Preps</b> </li>
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<ul>
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<li>None of the clones from which we extracted plasmids contained the correctly assembled insert. By PCR and diagnostic restriction digests on these plasmids we were able to distinguish two different misassembled versions of our desired Gibson Assembly reaction product. </li>
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<div class="pictext" style="height: 370px;">
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<div class="pictextl">Plasmid Digests<br>We did a second plasmid prep of our clones because the first weren’t very clear. Desmond figured out that by digesting the plasmids with EcoRV we ought to see a single 2kb band if pSB has closed on itself. We don't see anything like this in our clones, but we know from the rfp that this works (pSB-rfp is about 3kb, and contains a single EcoRV site in the pSB backbone). If the plasmid contains our gBlocks (or at least iGEMBLOCK 1 with aldA), then we expect to see two bands -  one at 1kb, a second at 5 or 6kb (depending whether we're looking at p450 or the adh plasmid). We don't see this either. We reckon that in most of the plasmids, some gBlocks have assembled in pSB1C3 but not iGEMBLOCK 1. In one of the clones (64) we see a band at 1kb but nothing else, so maybe iGEMBLOCK1 assembled in pSB, but not other gBlocks.
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<br><br>Interestingly, rfp looks spot-on while our results are similar to that in the last plasmid prep (everything slightly higher than 3kb, except 64). With rfp we expect a single band at 3kb (pSB is 2kb plus the rfp 'part' is 1 kb, and the whole construct contains a single EcoRV site in pSB). Notably, 64 was one of the small p450 colonies we patched from Gibson Assembly Product transformations.
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<a href="https://static.igem.org/mediawiki/2013/d/db/SydneyUniversity2013_results_plasmiddigest.jpg" rel="ibox" title="dhlB Plasmid Digest">
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<li> <b>PCR</b> </li>
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<li>We thought it could have been possible to assemble our entire pathway from smaller fragments salvaged from our Gibson Assembly reaction product. This proved impossible, presumably due to the extent of heterogenous template including both correctly and incorrectly assembled gBlocks in the Gibson Assembly reaction product. It may have been possible to do something similar using IDT’s gBlocks as template, however, this was not possible as we’d used up all of one of gBlocks during a second Gibson Assembly. </li>
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<div class="pictextl">GA Product PCR Assembly<br>Ap, Bp, Cp are from the p450 pathway, using our GA reaction product as template.  Aa, Ba and Ca are from the adh pathway. It looks like we can’t amplify what we want, or that it doesn’t exist in the GA reaction product.</div>
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<li> <b>Lessons</b> </li>
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<li> <b>Constitutive Expression</b> </li>
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<li>We suspect that some of the genes we tried to assemble <b>(eg, p450, Nishino et al, 2013, dropboxed in ‘reading’)</b> can harm the cells they're expressed in. If this is the case, then by a sort of natural screening we were only able to find colonies on plates that contain misassembled gBlocks that did not express these genes. </li>
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<li>Upon reflection, we approached the assembly of our pathway with an almost child-like ignorance and optimism. Our promoter was specifically designed to maximise expression of our construct, as if ‘the more pollutant degrading genes, the better’. If the hypothesis above is correct, then we might have had more success with an inducible promoter.</li>
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<li> <b>Modularity</b> </li>
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<li> Consider the parable of the watchmakers: <br><br> There once were two watchmakers, named Hora and Tempus, who manufactured very fine watches. Both of them were highly regarded, and the phones in their workshops rang frequently - new customers were constantly calling them. However, Hora prospered, while Tempus became poorer and poorer and ?nally lost his shop.What was the reason?<br><br>The watches the men made consisted of about 1,000 parts each. Tempus had so constructed his that if he had one partly assembled and had to put it down - to answer the phone say - it immediately fell to pieces and had to be reassembled from the elements. The better the customers liked his watches, the more they phoned him, the more difficult it became for him to find enough uninterrupted time to finish a watch.<br><br>The watches that Hora made were no less complex than those of Tempus. But he had designed them so that he could put together subassemblies of about ten elements each. Ten of these subassemblies, again, could be put together into a larger subassembly; and a system of ten of the latter sub-assemblies constituted the whole watch. Hence, when Hora had to put down a partly assembled watch in order to answer the phone, he lost only a small part of his work, and he assembled his watches in only a fraction of the man-hours it took Tempus.<br><br>H.A. Simon, The Architecture of Complexity, 1962.</li>
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<li>The sequences we had synthesised as gBlocks by IDT were designed so that they could only be assembled in the whole DCA-degradation pathway, rather than as parts within pSB1C3 which could then be assembled piece-by-piece. This meant that our success relied on the correct assembly of the entire pathway, and when this failed, that we were unable to access parts of the pathway (without PCR assembly, or ordering new, complementary gBlocks).</li>
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<li> <b>Plans</b> </li>
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<li> <b>Replacement of gBlocks</b> </li>
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<li>By replacing a single <a href="https://2013.igem.org/Team:SydneyUni_Australia/Project/Design">gBlock</a>, it might be possible to find correctly assembled Gibson products by substituting our strong constitutive promoter Psyn with a repressible promoter.</li>
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<li>With four new gBlocks, it might be possible to assemble some of the important genes in our pathway (aldA, p450, adh1b1) in a BioBrick vector, so that they could then be subsequently assembled piecewise. </li>
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</ul>
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<li> <b>PCR Assembly</b> </li>
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<ul>
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<li>It might be possible to PCR amplify genes directly from our gBlocks as template rather than our Gibson Assembly reaction product. Alternatively, with the design of new primers, the genes of interest might be amplified from gBlocks for cloning into a BioBrick vector. With either of these options, sequence fidelity may be an issue.</li>
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<div class="unlink">Assembly of dhlB-dhlA in pSB1C3</div>
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<ul>
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<li>While struggling with Gibson Assembly of our whole pathway we turned to the extraction, cloning and characterisation of two parts in our pathway, dhlB and dhlA <b>(link to pathway or pic)</b>. These two genes had been cloned into pUC19 by others in our lab <b>(WHO WAS IT, pretty sure Jake and Deb?)</b>.</li>
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<li><b>Amplification</b></li>
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<li>We designed primers specifically for amplifying dhlB and dhlA, while removing a forbidden EcoRI site between them. Design with the primers allowed us to try cloning each gene by itself and also together into the shipping vector pSB1C3.</li>
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<div class="pictext">
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* Early in 2013 it was discovered that a monooxygenase from a Polaromonas sp. (JS666) was responsible for the initial steps of DCE and DCA degradation by heterologous expression in E. Coli ([http://aem.asm.org/content/79/7/2263.short) Nishino et al, 2013]). As far as we know, this enzyme effectively substitutes for ToMO in our pathway. Due to the shorter length of the three-gene p450 complex (~3kb) we decided to synthesise this enzyme instead of ToMO.
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<div class="pictextl"><b>DESCRIPTION</b></div>
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* The strain that carries this enzyme was first isolated from chloroethene contaminated sites by our primary supervisor, Nick Coleman, in the Stone Age ([http://aem.asm.org/content/68/6/2726.full Coleman et al, 2002]).  
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<div class="pictextl">PCR Junction Screening of <a href="http://parts.igem.org/Part:BBa_K1115008">AB22</a></div>
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<a href="https://static.igem.org/mediawiki/2013/c/c7/SydneyUniversity2013_results_junctionscreen.jpg" rel="ibox" title="ToMO Cl- Assay">
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'''adh1b2, human liver alcohol dehydrogenase'''
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<li><b> Cloning </b></li>
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<ul>
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<div class="pictext">
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* Human liver alcohol dehydrogenases have been shown to be active on a broad range of substrates, including haloalcohols and haloaldehydes ([http://pubs.acs.org/doi/pdf/10.1021/bi00870a034 Blair & Vallee, 1966]). They’ve also been expressed in E. Coli before (eg, [http://onlinelibrary.wiley.com/doi/10.1111/j.1530-0277.1993.tb00849.x/abstract Zheng et al, 1993]) using cDNA, but we were able to use a modified mRNA sequence from GenBank ([http://www.ncbi.nlm.nih.gov/nuccore/BC033009 BC033009.2]). We were also able to choose between many types and classes of human liver enzyme, so we picked the one with the greatest turnover on ethanol as substrate (Edenberg, 2007).
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<div class="pictextl">We cloned the PCR fragments into pSB1C3 and transformed the ligation product. We were greatly assisted by ligating into a <a href="http://parts.igem.org/Part:BBa_J04450">BBa_J04450</a>, extracted from the <a href="http://parts.igem.org/Help:Protocols/Linearized_Plasmid_Backbones">linearised plasmid pSB1C3</a> in the Distribution Kit, which provided a neat red-white screen.</div>
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<a href="https://static.igem.org/mediawiki/2013/5/55/SydneyUni2013_Results_Assembly_Cloning_Screen.jpg" rel="ibox" title="ToMO Cl- Assay">
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<li>After PCR screening of the junctions between our parts and pSB1C3, we extracted the plasmids from a few that looked OK for further confirmation and submission to the iGEM HQ.</li>
 
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<div class="pictext">
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=== '''Order of gene expression'''===
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<div class="pictextl"><b>DESCRIPTION</b></div>
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<div id="gblocks"></div>
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* We chose to have a single, strong constitutive promoter in our pathway (see below, promoter), so it made sense to place our genes in order from last to first in order that at every step in the pathway there would be an excess of the enzyme required for the substrate, thus minimising the build-up of toxic intermediates.  
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<center>
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* However, chloroacetaldehyde appears to be the most toxic of the intermediates produced while DCA is degraded (Janssen et al, 1994). It seemed necessary to position the enzyme specific to aldA immediately after the promoter to ensure that chloroacetaldehyde was rapidly removed from the cell.  
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<li><b> Characterisation of submitted parts with the constitutive promoter Pcat </b></li>
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<ul>
+
-
<div class="pictext" style="height: 150px">
+
-
<div class="pictextl">Colourmetric assay of chloride release from dhlB activity on chloroacetate (in blue) and dhlA activity on 1,2-Dichloroethane (1,2-DCA, in red). Standard curve generated with 0.0, 0.1, 0.2, 0.5, 1.0, 1.5, and 2.0mM NaCl in KP buffer. TOP10 E.coli cells were harvested at OD600=0.4, pelleted and washed three times in KP buffer.  Cells were resuspended in 2mM chloroacetate or 1,2-DCA and incubated for 16hrs at 37°C and 200rpm. Cells were then pelleted and assayed using the <a href="https://2013.igem.org/Team:SydneyUni_Australia/Project/Protocols Bergmann and Sanik">chloride assay</a> and the absorbance at 460nm read. Data for each condition is in triplicate (standard deviation <0.26 Cl- (mM)). </div>
+
-
<div class="pictextr">
+
-
<center>
+
-
<a href="https://static.igem.org/mediawiki/2013/b/b5/Cl_assay_Graph_with_bars.png" rel="ibox" title="ToMO Cl- Assay">
+
-
<img src="https://static.igem.org/mediawiki/2013/b/b5/Cl_assay_Graph_with_bars.png" height="100">
+
-
</a>
+
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</center>
+
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</div>
+
-
</div>
+
-
<br><br>
+
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<div class="pictext" style="height: 150px">
+
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<div class="pictextl">Chloride release from dhlB activity on chloroacetate (in blue) and dhlA activity on 1,2-Dichloroethane (1,2-DCA, in red). Negative Control was BBa_K1115008, the promoterless dhlB-dhlA coding region, <a href="http://parts.igem.org/Part:BBa_K1115009">BBa_K1115009</a> is dhlB-dhlA constitutively expressed by PCat (<a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_I14033">BBa_I14033]</a>), <a href="http://parts.igem.org/Part:BBa_K1115010">BBa_K1115009</a> is constitutively expressed by PTet (<a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_R0040">BBa_R0040]</a>, and the positive control is the Coleman lab pUC19 house plasmid expressing dhlB-dhlA with the same RBS.</div>
+
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<div class="pictextr">
+
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<center>
+
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<a href="https://static.igem.org/mediawiki/2013/0/0a/Percentage_degradation.png" rel="ibox" title="ToMO Cl- Assay">
+
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<img src="https://static.igem.org/mediawiki/2013/0/0a/Percentage_degradation.png" height="100">
+
-
</a>
+
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</center>
+
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</div>
+
-
</div>
+
-
+
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<li>Estimated percentage degradation of chloroacetate and 1,2-DCA as determined by: <br><br>
+
-
<center><img src="https://static.igem.org/mediawiki/2013/6/6f/Percentage_degradation_equation.png"></center><br><br>
+
-
Where the Test cell supernatant is from BBa_K1115009 or BBa_K1115010, the Negative cell supernatant is from BBa_K1115008, and the substrate concentration is 2mM Chloroacetate or 1,2-DCA. Note that values of over 100% degradation are misleading and likely indicate endogenous Cl- production (i.e. Cl- production from other cellular processes unrelated to target substrate metaboloism.
+
-
</li>
+
-
+
-
</ul>
+
-
</ul>
+
-
</ul>
+
-
<div class="unlink">Constitutive Expression of dhlB-dhlA, Degradation of DCA and Chloroacetate</div>
+
-
<ul>
+
-
<li>After sending dhlB and dhlA to iGEM HQ, we began characterisation by cloning a constitutive promoter (<a href="http://parts.igem.org/Part:BBa_I14033">BBa_I14033</a>) from the Distribution Kit in front of our parts.</li>
+
-
<li>Amplification</li>
+
-
<ul>
+
-
<li>Pcat ([http://parts.igem.org/Part:BBa_I14033 BBa_I14033]) is 38bp, but with our primers produced a 280bp fragment, 4th well from left.</li>
+
-
+
-
<div class="pictext"0 style="height:300px;">
+
[[File:Sydney_Australia_2013_Design_orderofgeneexpression.jpg|centre]]
-
<div class="pictextl">The Gel demonstrating Pcat Amplification:<br>1.0% agarose Gel of PCR products from Distribution Kit: Loading order of was 1kb ladder (from the top: 10, 8, 6, 5, 4, 3, 2, 1.5, 1, 0.5kb), LacI generator PCR product (BBa_P0412 template), 100bp ladder (100, 200, 300, 400, 500, 6000, 700, 800, 900, 1200, 1500bp) PCat (BBa_I14033 template) and PLac. The band for PCat indicates the correct length including ends of pSB1C: 38bp + ~250bp=~290bp.
+
-
<br>
+
-
The PCat PCR products were combined (400uL) and column purified using the QiaQuick Kit (see Protocols tab). As we were attempting to construct an inducible system with PCat constitutively promoting the LacI generator and we had multiple LacI PCR products, we attempted a gel band extraction of the correct band but were unsuccessful. 
+
-
<br>
+
-
Having lost our repressor part, we digested PCat with EcoRI and SpeI, and our dhlB-dhlA part BBa_K1115008 with EcoRI and XbaI at 37oC. Digested DNA was purified, mixed and ligated for one hour at room temperature to form a putative construct BBa_K1115009 which was transformed to chemically competent TOP10 cells.
+
-
</div>
+
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<div class="pictextr">
+
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<center>
+
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<a href="https://static.igem.org/mediawiki/2013/1/1d/SydneyUniversity2013_results_Pcatamplification.jpg" rel="ibox" title="Pcat Amplification">
+
-
<img src="https://static.igem.org/mediawiki/2013/1/1d/SydneyUniversity2013_results_Pcatamplification.jpg" height="100">
+
-
</a>
+
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</center>
+
-
</div>
+
-
</div>
+
-
+
-
<li>Ptet ([http://parts.igem.org/Part:BBa_R0040 BBa_R0040])</b> is ~50bp, but with our primers produced fragment ~300bp, 1st well from left.</li>
+
-
<div class="pictext">
+
-
<div class="pictextl">The Gel demonstrating Ptet Amplification:</div>
+
-
<div class="pictextr">
+
-
<center>
+
-
<a href="https://static.igem.org/mediawiki/2013/f/f3/SydneyUniversity2013_results_Ptetamplification.jpg" rel="ibox" title="Ptet Amplification">
+
-
<img src="https://static.igem.org/mediawiki/2013/f/f3/SydneyUniversity2013_results_Ptetamplification.jpg" height="100">
+
-
</a>
+
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</center>
+
-
</div>
+
-
</div>
+
-
</ul>
+
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<li>Phenotypic Assays</li>
+
-
<ul>
+
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<li>After <a href="https://2013.igem.org/Team:SydneyUni_Australia/Project/Protocols">cloning</a> into a plasmid containing dhlB and dhlA, we screened for clones expressing our construct. We made <a href="https://2013.igem.org/Team:SydneyUni_Australia/Project/Protocols">screening plates</a> (contained LB-agar-chloramphenicol- 10mMchloroacetate-phenol red at pH 6.8) that allowed us to pick clones that looked like they were successfully expressing one of our genes of interest.</li>
+
-
<div class="pictext">
+
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<div class="pictextl">Screening plates that allowed us to isolate clones</div>
+
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<div class="pictextr">
+
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<center>
+
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<a href="https://static.igem.org/mediawiki/2013/3/35/SydneyUniversity2013_results_screeningplates.jpg" rel="ibox" title="Screening Plates">
+
-
<img src="https://static.igem.org/mediawiki/2013/3/35/SydneyUniversity2013_results_screeningplates.jpg" height="100">
+
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</a>
+
-
</center>
+
-
</div>
+
-
</div>
+
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<li>We showed degradation of chloroacetate and DCA by chloride assay. </li>
+
-
<div class="pictext">
+
-
<div class="pictextl">Colourmetric assay of chloride release: Cl assay Graph with standard curve</div>
+
-
<div class="pictextr">
+
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<center>
+
-
<a href="https://static.igem.org/mediawiki/parts/8/89/Cl_assay_Graph_with_standard_curve.png" rel="ibox" title="Chloride Assay">
+
-
<img src="https://static.igem.org/mediawiki/parts/8/89/Cl_assay_Graph_with_standard_curve.png" height="100">
+
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</a>
+
-
</center>
+
-
</div>
+
-
</div>
+
-
</ul>
+
-
+
-
+
-
</ul>
+
-
</div>
+
-
</html>
+
 +
 +
 +
=== '''Optimising a promoter'''===
 +
 +
* We used a single synthetic promoter region based on Plac, but including changes that other people have shown increase RNA polymerase binding at the promoter region. All the changes were made to maximise RNA polymerase recognition.
 +
 +
[[File:Sydney_Australia_2013_Design_promoterregion.jpg|centre]]
 +
 +
 +
=== '''Ribosome binding sites'''===
 +
 +
We added or modfied the same RBS (AAGGAGG) before all our genes, for a mix of these reasons:
 +
*The natural sequences we borrowed were from diverse hosts and didn’t contain RBS optimal for E. coli.
 +
*Sequences were found as annotated coding regions without an RBS.
 +
*Sequences had apparently sub-optimal RBSs too close or distant to the start codon.
 +
 +
 +
=== '''Restriction sites'''===
 +
 +
We removed all restriction sites in our sequences that are forbidden within Registry parts (EcoRI, NotI, XbaI, SpeI, PstI). This had to be done while ensuring not to change codon usage, or change to an rare codon. We used this  [http://www.kazusa.or.jp/codon/cgi-bin/showcodon.cgi?species=83333&aa=11&style=GCG codon table].
 +
 +
 +
=== '''Codon usage'''===
 +
 +
An online [http://nihserver.mbi.ucla.edu/RACC/ application] was used to identify codons rarely used by E. Coli. Again, changes had to be made while ensuring not to change codon usage. All stop codons were converted to the more common variant and included as double-stops (TAATAA).
 +
 +
 +
=== '''GC content and hairpins'''===
 +
 +
*The template we used for Gibson Assembly was purchased as gBlocks from IDT with their iGEM discount.
 +
*Due to the assembly of larger fragments (~500bp) from smaller oligos (60-120bp) during gene synthesis, some regions need to be avoided. These are regions contain extremes of GC content and regions that form small, six-base hairpins. We tried to avoid problems by running all our sequences through the [http://www.idtdna.com/order/gblockwizard.aspx gBlocks wizard] before ordering, and addressing any problems. However, the application failed to find a few problematic regions and these caused delay before the arrival of our gBlocks.
 +
 +
 +
== '''References'''==
 +
 +
*Blair, A. H., & Vallee, B. L. (1966). Some Catalytic Properties of Human Liver Alcohol Dehydrogenase*. Biochemistry, 5(6), 2026-2034.
 +
*Edenberg, H. J. (2007). The genetics of alcohol metabolism: role of alcohol dehydrogenase and aldehyde dehydrogenase variants. Alcohol Research & Health.
 +
*Giacomini, Alessio, et al. "Construction of multipurpose gene cartridges based on a novel synthetic promoter for high-level gene expression in Gram-negative bacteria." Gene 144.1 (1994): 17-24.
 +
* Janssen, D. B., Keuning, S., & Witholt, B. (1987). Involvement of a quinoprotein alcohol dehydrogenase and an NAD-dependent aldehyde dehydrogenase in 2-chloroethanol metabolism in Xanthobacter autotrophicus GJ10. Journal of general microbiology, 133(1), 85-92.
 +
*Janssen, D. B., Pries, F., & Van der Ploeg, J. R. (1994). Genetics and biochemistry of dehalogenating enzymes. Annual Reviews in Microbiology, 48(1), 163-191.
 +
*Khairnar, N. P., Misra, H. S., & Apte, S. K. (2003). Pyrroloquinoline–quinone synthesized in< i> Escherichia coli</i> by pyrroloquinoline–quinone synthase of< i> Deinococcus radiodurans</i> plays a role beyond mineral phosphate solubilization. Biochemical and biophysical research communications, 312(2), 303-308.
 +
*Liu, Mofang, et al. "A mutant spacer sequence between-35 and-10 elements makes the Plac promoter hyperactive and cAMP receptor protein-independent."Proceedings of the National Academy of Sciences of the United States of America 101.18 (2004): 6911-6916.
 +
*Ross, Wilma, et al. "Escherichia coli promoters with UP elements of different strengths: modular structure of bacterial promoters." Journal of bacteriology180.20 (1998): 5375-5383.
 +
*Vardar, G., & Wood, T. K. (2005). Protein engineering of toluene-o-xylene monooxygenase from Pseudomonas stutzeri OX1 for enhanced chlorinated ethene degradation and o-xylene oxidation. Applied microbiology and biotechnology, 68(4), 510-517.
 +
*Zheng, C. F., Wang, T. T., & Weiner, H. (1993). Cloning and Expression of the Full‐Length cDNAS Encoding Human Liver Class 1 and Class 2 Aldehyde Dehydrogenase. Alcoholism: Clinical and Experimental Research, 17(4), 828-831.
{{Team:SydneyUni_Australia/Footer}}
{{Team:SydneyUni_Australia/Footer}}

Revision as of 04:17, 28 September 2013

SydneyUniversity Top Banner.jpg SydneyUniversity Bottom Banner.jpg


Gibson

Gibson Assembly *should* make for a much easier, simpler, rapid assembly of different genes than conventional PCR and cloning, plus there’s much more flexibility for optimisation through gene synthesis.

  • The assembly works on fragments of DNA with ~30bp of overlapping sequence, which is exposed as 5’ single-stranded overhangs by an exonuclease. A ligase joins the overlapping regions and a polymerase fills in any gaps left by the exonuclease. These enzymes can all work together in a single reaction tube with many different overlapping fragments, making the assembly a very, very simple activity. Gibson Assembly is based on the older technique of [http://nar.oxfordjournals.org/content/32/12/e98.full PCR Assembly], with the similar reliance on the initial construction of 200+bp fragments from smaller oligos, but with a greater degree of sequence fidelity due to less polymerase activity.
  • Here’s a great introduction from IDT’s magazine, [http://www.idtdna.com/pages/decoded/decoded-articles/core-concepts/decoded/2012/01/10/isothermal-assembly-quick-easy-gene-construction DECODED], and a more in-depth webinar.
  • If you’re historically-minded or want more detail, try the [http://diyhpl.us/~bryan/papers2/bio/venter/Enzymatic%20assembly%20of%20DNA%20molecules%20up%20to%20several%20hundred%20kilobases.pdf original paper] in which Gibson Assembly was described - or one of the coolest and most famous applications of Gibson, building a [http://www.ncbi.nlm.nih.gov/books/NBK84435/ synthetic genome].

Design

Choice of genes

Mox, chloroethanol dehydrogenase

  • In sketches of our project (February-March), we planned to use Mox as the alcohol dehydrogenase converting chloroethanol to chloroacetaldehyde. After a little more research we discovered that this enzyme requires the co-factor PQQ, and unfortunately for us, this co-factor requires many genes for its synthesis, which would have made our constructs too complex (Khairnar et al, 2003).
  • Mox had seemed like an obvious choice because it would be sourced from Xanthobacter autotrophicus GJ10, the most well-documented DCA-degrader ([http://mic.sgmjournals.org/content/133/1/85.full.pdf Janssen et al, 1987])

aldA, chloroacetaldehyde dehydrogenase, dhlB, haloacid dehalogenase, and dhlA, haloalkane dehalogenase

  • We used a sequence for aldA from Xanthobacter autrophicus GJ10, but used sequences for dhlB and dhlA from a different strain, Xanthobacter autrophicus EL4, which was isolated and characterised in the Coleman lab. These two genes had been identified and cloned into pUC19 by Jake Munro, a research assistant in the Coleman lab. When our Gibson Assembly ran into problems, we continued to work with dhlB and dhlA from EL4 in our lab and submitted these two genes as parts.
  • These genes are shared by a few different species of bacteria that degrade DCA (Janssen et al, 1994), and most have been conventionally characterised by extraction and heterologous expression of a single gene at a time.

ToMO, toluene-o-xylene monooxygenase

  • Early in our project we showed that ToMO can degrade DCA. This would not only eliminate the need for something like PQQ-synthase, but also make for a shorter, less complex pathway involving just 3 enzymes (a monooxygenase, dehydrogenase and dehalogenase).
  • We initially planned to synthesise ToMO, allowing us to remove forbidden restriction sites in the process (EcoRI and PstI). However, due to the sheer size of the monooxygenase cluster (~5kb) we could not afford to have this gene and all of our others synthesised.
  • The gene we worked with initially has been extensively used for protein engineering by [http://fenske.che.psu.edu/faculty/wood/group/publications/pdf/ToMO%20TCE%20mutagenesis%20Vardar[1].pdf Varder & Wood (2005)].

p450, cytochrome p450 monooxygenase

  • Early in 2013 it was discovered that a monooxygenase from a Polaromonas sp. (JS666) was responsible for the initial steps of DCE and DCA degradation by heterologous expression in E. Coli ([http://aem.asm.org/content/79/7/2263.short) Nishino et al, 2013]). As far as we know, this enzyme effectively substitutes for ToMO in our pathway. Due to the shorter length of the three-gene p450 complex (~3kb) we decided to synthesise this enzyme instead of ToMO.
  • The strain that carries this enzyme was first isolated from chloroethene contaminated sites by our primary supervisor, Nick Coleman, in the Stone Age ([http://aem.asm.org/content/68/6/2726.full Coleman et al, 2002]).

adh1b2, human liver alcohol dehydrogenase

  • Human liver alcohol dehydrogenases have been shown to be active on a broad range of substrates, including haloalcohols and haloaldehydes ([http://pubs.acs.org/doi/pdf/10.1021/bi00870a034 Blair & Vallee, 1966]). They’ve also been expressed in E. Coli before (eg, [http://onlinelibrary.wiley.com/doi/10.1111/j.1530-0277.1993.tb00849.x/abstract Zheng et al, 1993]) using cDNA, but we were able to use a modified mRNA sequence from GenBank ([http://www.ncbi.nlm.nih.gov/nuccore/BC033009 BC033009.2]). We were also able to choose between many types and classes of human liver enzyme, so we picked the one with the greatest turnover on ethanol as substrate (Edenberg, 2007).


Order of gene expression

  • We chose to have a single, strong constitutive promoter in our pathway (see below, promoter), so it made sense to place our genes in order from last to first in order that at every step in the pathway there would be an excess of the enzyme required for the substrate, thus minimising the build-up of toxic intermediates.
  • However, chloroacetaldehyde appears to be the most toxic of the intermediates produced while DCA is degraded (Janssen et al, 1994). It seemed necessary to position the enzyme specific to aldA immediately after the promoter to ensure that chloroacetaldehyde was rapidly removed from the cell.


Sydney Australia 2013 Design orderofgeneexpression.jpg


Optimising a promoter

  • We used a single synthetic promoter region based on Plac, but including changes that other people have shown increase RNA polymerase binding at the promoter region. All the changes were made to maximise RNA polymerase recognition.
Sydney Australia 2013 Design promoterregion.jpg


Ribosome binding sites

We added or modfied the same RBS (AAGGAGG) before all our genes, for a mix of these reasons:

  • The natural sequences we borrowed were from diverse hosts and didn’t contain RBS optimal for E. coli.
  • Sequences were found as annotated coding regions without an RBS.
  • Sequences had apparently sub-optimal RBSs too close or distant to the start codon.


Restriction sites

We removed all restriction sites in our sequences that are forbidden within Registry parts (EcoRI, NotI, XbaI, SpeI, PstI). This had to be done while ensuring not to change codon usage, or change to an rare codon. We used this [http://www.kazusa.or.jp/codon/cgi-bin/showcodon.cgi?species=83333&aa=11&style=GCG codon table].


Codon usage

An online [http://nihserver.mbi.ucla.edu/RACC/ application] was used to identify codons rarely used by E. Coli. Again, changes had to be made while ensuring not to change codon usage. All stop codons were converted to the more common variant and included as double-stops (TAATAA).


GC content and hairpins

  • The template we used for Gibson Assembly was purchased as gBlocks from IDT with their iGEM discount.
  • Due to the assembly of larger fragments (~500bp) from smaller oligos (60-120bp) during gene synthesis, some regions need to be avoided. These are regions contain extremes of GC content and regions that form small, six-base hairpins. We tried to avoid problems by running all our sequences through the [http://www.idtdna.com/order/gblockwizard.aspx gBlocks wizard] before ordering, and addressing any problems. However, the application failed to find a few problematic regions and these caused delay before the arrival of our gBlocks.


References

  • Blair, A. H., & Vallee, B. L. (1966). Some Catalytic Properties of Human Liver Alcohol Dehydrogenase*. Biochemistry, 5(6), 2026-2034.
  • Edenberg, H. J. (2007). The genetics of alcohol metabolism: role of alcohol dehydrogenase and aldehyde dehydrogenase variants. Alcohol Research & Health.
  • Giacomini, Alessio, et al. "Construction of multipurpose gene cartridges based on a novel synthetic promoter for high-level gene expression in Gram-negative bacteria." Gene 144.1 (1994): 17-24.
  • Janssen, D. B., Keuning, S., & Witholt, B. (1987). Involvement of a quinoprotein alcohol dehydrogenase and an NAD-dependent aldehyde dehydrogenase in 2-chloroethanol metabolism in Xanthobacter autotrophicus GJ10. Journal of general microbiology, 133(1), 85-92.
  • Janssen, D. B., Pries, F., & Van der Ploeg, J. R. (1994). Genetics and biochemistry of dehalogenating enzymes. Annual Reviews in Microbiology, 48(1), 163-191.
  • Khairnar, N. P., Misra, H. S., & Apte, S. K. (2003). Pyrroloquinoline–quinone synthesized in< i> Escherichia coli</i> by pyrroloquinoline–quinone synthase of< i> Deinococcus radiodurans</i> plays a role beyond mineral phosphate solubilization. Biochemical and biophysical research communications, 312(2), 303-308.
  • Liu, Mofang, et al. "A mutant spacer sequence between-35 and-10 elements makes the Plac promoter hyperactive and cAMP receptor protein-independent."Proceedings of the National Academy of Sciences of the United States of America 101.18 (2004): 6911-6916.
  • Ross, Wilma, et al. "Escherichia coli promoters with UP elements of different strengths: modular structure of bacterial promoters." Journal of bacteriology180.20 (1998): 5375-5383.
  • Vardar, G., & Wood, T. K. (2005). Protein engineering of toluene-o-xylene monooxygenase from Pseudomonas stutzeri OX1 for enhanced chlorinated ethene degradation and o-xylene oxidation. Applied microbiology and biotechnology, 68(4), 510-517.
  • Zheng, C. F., Wang, T. T., & Weiner, H. (1993). Cloning and Expression of the Full‐Length cDNAS Encoding Human Liver Class 1 and Class 2 Aldehyde Dehydrogenase. Alcoholism: Clinical and Experimental Research, 17(4), 828-831.

With thanks to:



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