Team:Heidelberg/Delftibactin/DelH

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                                   <h1>Week 2</h1>
                                   <h1>Week 2</h1>
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                                   <p style="font-size:10pt; text-align:justify; position:relative; margin-left:6%;">Using the designed primers DelH_f1_PacI_fw and DelH_f1_SalI_rev, the first PCRs to amplify DelH F1 as well as DelH_f2_SalI_fw and DelH_f2_KpnI_rev to amplify DelH F2 were performed and conditions optimized. Additionally, necessary backbone fragments pSB6A1 and lacZ were restriction digested. New BioBricks to obtain the AraC promotor (I13453 and K206000) were chosen.  
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                                  <p>At the beginning of the week, we could verify that the Gibson Assembly for Tripeptide I was indeed positive, however, the other Gibson Assemblies did not work properly. Instead of picking new colonies, we decided to optimize the Gibson recipe instead, as backbone religations were the most common problem. With these improved protocols, we used Gibson Assembly for the Dipeptide, Tripeptide II and Tetrapeptide I, later that week, Tetrapeptide II followed. After the Transformation to DH10β cells and screening by restriction digest we could send samples for the Dipeptide and Tetrapeptide I to sequencing and obtained a positive alignment. Hence we transformed BAP I cells with the positive constructs. The same was...
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                                   <h1>Week 3</h1>
                                   <h1>Week 3</h1>
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                                   <p style="font-size:10pt; text-align:justify; position:relative; margin-left:6%;">Since the reverse backbone primer DN08:AraCbb_PacI_rev did not work, a new one (DN06:AraCbb_PacI_rev2) was ordered, together with the colony PCR screening primer DN07:Screen_DelH_rev and DN13:Screen_DelH_fw to check for correct ligation of the DelH fragment F1.  
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                                  <p>At the beginning of the week, we could verify that the Gibson Assembly for Tripeptide I was indeed positive, however, the other Gibson Assemblies did not work properly. Instead of picking new colonies, we decided to optimize the Gibson recipe instead, as backbone religations were the most common problem. With these improved protocols, we used Gibson Assembly for the Dipeptide, Tripeptide II and Tetrapeptide I, later that week, Tetrapeptide II followed. After the Transformation to DH10β cells and screening by restriction digest we could send samples for the Dipeptide and Tetrapeptide I to sequencing and obtained a positive alignment. Hence we transformed BAP I cells with the positive constructs. The same was...
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                                   <h1>Week 4</h1>
                                   <h1>Week 4</h1>
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                                   <p style="font-size:10pt; text-align:justify; position:relative; margin-left:6%;">The elongation of the pSB6A1-AraC-lacZ backbone urned out to be troublesome. So in week 4, we performed different restriction digests with our final backbone pSB6A1-AraC-lacZ as well as with the former construct pSB1C3-AraC-lacZ to ensure identity of the backbone. Besides, the amplification of the DelH fragment F1 was planned: It is amplified divided in 2 subfragments - fragment F1a and fragment F1b. Each one of the fragments has the size of 5 Kb. </p>
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                                   <h1>Week 5</h1>
                                   <h1>Week 5</h1>
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                                   <p style="font-size:10pt; text-align:justify; position:relative; margin-left:6%;">At the beginning of the week, we could verify that the Gibson Assembly for Tripeptide I was indeed positive, however, the other Gibson Assemblies did not work properly. Instead of picking new colonies, we decided to optimize the Gibson recipe instead, as backbone religations were the most common problem. With these improved protocols, we used Gibson Assembly for the Dipeptide, Tripeptide II and Tetrapeptide I, later that week, Tetrapeptide II followed.</p>
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                                   <p style="font-size:10pt; text-align:justify; position:relative; margin-left:6%;">In this week, we started all over again to assemble the backbone pSB6A1-AraC-lacZ: digesting AraC, lacZ and PSB6A1 based on the precedently amplified fragments. The amplification of DelH was continued and we succesfully amplified all 3 fragments and gel extracted them. </p>
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                                   <h1>Week 6</h1>
                                   <h1>Week 6</h1>
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                                   <p style="font-size:10pt; text-align:justify; position:relative; margin-left:6%;">Because we amplified all DelH-fragments needed and assembled the backbone in week 5, this week, we assembled the final plasmid pHM01. Therefore, every fragment was digested with two distinct enzymes, and then ligated. The ligated plasmid pHM01 was purified and electroporated in two separate DH10ß aliquots. Unfortunately, the screening via colony-PCR was negative, so none of the transformed E.coli successfully received the plasmid. </p>
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                                   <h1>Week 7</h1>
                                   <h1>Week 7</h1>
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                                   <p style="font-size:10pt; text-align:justify; position:relative; margin-left:6%;">In week 7, we reamplified the DelH fragments used for the transformation in week 6. We found out that amplification of fragments F1a and F1b was not reproducible. As conclusion, we designed new primers for DelH, which are capable to amplify the beginning of DelH in a more efficient and specific manner. The overview summarizes the primers we ordered and their performance in amplificating DelH F1a. Primer DN11 worked really well and is used in the next experiments. </p>
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                                   <h1>Week 8</h1>
                                   <h1>Week 8</h1>
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                                   <p style="font-size:10pt; text-align:justify; position:relative; margin-left:6%;">After improving the amplification of the beginning of DelH (fragment 1a with the primer DN11), we used a higher concentration in the ligation assembling the pHM01 plasmid. The transformation of DH10ß cells was performed with electroporation. Four colonies were positive in the screening-PCR, but did not express lacZ (no blue color). To quantify if DelH is possibly expressed anyhow, we plan a SDS-PAGE and induce the bacteria with higher concentrations of arabinose and X-Gal.
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                                       <h1>Week 9</h1>
                                       <h1>Week 9</h1>
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                                       <p style="font-size:10pt; text-align:justify; position:relative; margin-left:6%;">The SDS-PAGE showed no clear band at ~600 kDa, so we conclude that there is most probably no DelH expression in the analyzed colonies. To confirm the result, a restriction digest was performed. The colonies did not show the expected pattern, so they indeed did not contain the desired plasmid. To perform a new assembly of the plasmid pHM01, the fragments of DelH and the backbone had to be reamplified. The amplifications of the DelH fragments went well, but the backbone caused again some difficulties and it was not possible to amplify it.  
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In order to transfer the gold precipitating NRPS from <i>D. acidovorans</i> to <i>E.coli</i>, the necessary modules will be amplified from the D. acidovorans genome and assembled as plasmids. Due to its large size of 18 Kb, the module DelH will be expressed on a separate plasmid. A strategy was developed, primers designed accordingly and necessary BioBricks obtained from the distribution. The <i>D. acidovorans</i> was obtained from the DSMZ and cultured in Acidovorax complex medium.  
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                                   <h1>Week 10</h1>
                                   <h1>Week 10</h1>
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                                   <p style="font-size:10pt; text-align:justify; position:relative; margin-left:6%;">When rechecking all amplified fragments, we did not find what we expected. On the one hand, the concentrations of the fragments were too low and could not be seen on the gel. On the other hand, a re-amplification of the fragments with the appropriate primers was not successful. Additionally, a new polymerase (Phusion Flash) was used. Interestingly for the DelH 1b fragment, the fragment amplified from the genomic DNA was slightly larger than the amplified fragments from itself.  
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                                  <p>At the beginning of the week, we could verify that the Gibson Assembly for Tripeptide I was indeed positive, however, the other Gibson Assemblies did not work properly. Instead of picking new colonies, we decided to optimize the Gibson recipe instead, as backbone religations were the most common problem. With these improved protocols, we used Gibson Assembly for the Dipeptide, Tripeptide II and Tetrapeptide I, later that week, Tetrapeptide II followed. After the Transformation to DH10β cells and screening by restriction digest we could send samples for the Dipeptide and Tetrapeptide I to sequencing and obtained a positive alignment. Hence we transformed BAP I cells with the positive constructs. The same was...
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                                   <h1>Week 11</h1>
                                   <h1>Week 11</h1>
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                                   <p style="font-size:10pt; text-align:justify; position:relative; margin-left:6%;">Based on this week's experiments and in silico analysis to validate the fragments DelH F1a, F1b and F2, we discarded the restriction digest and ligation strategy for the assembly of the DelH plasmid. Instead, we chose Gibson assembly as alternative method, developed a strategy and designed primers accordingly. We are positive, to finally assemble pHM02.  
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                                  <p>At the beginning of the week, we could verify that the Gibson Assembly for Tripeptide I was indeed positive, however, the other Gibson Assemblies did not work properly. Instead of picking new colonies, we decided to optimize the Gibson recipe instead, as backbone religations were the most common problem. With these improved protocols, we used Gibson Assembly for the Dipeptide, Tripeptide II and Tetrapeptide I, later that week, Tetrapeptide II followed. After the Transformation to DH10β cells and screening by restriction digest we could send samples for the Dipeptide and Tetrapeptide I to sequencing and obtained a positive alignment. Hence we transformed BAP I cells with the positive constructs. The same was...
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                                   <h1>Week 12</h1>
                                   <h1>Week 12</h1>
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                                   <p style="font-size:10pt; text-align:justify; position:relative; margin-left:6%;">The primers arrived and we started amplifying the Gibson fragments using different approaches. Since G1 could not be PCR amplified, we decided to divide G1 into sub fragments G1a and G1b, for which we had designed and ordered primers already. The amplification of fragment G2 worked well and was produced in reasonable amounts. Alternatively, we also produced the entire DelH as one fragment G0, as well as further sub fragments DelH G1/2a, G1b/2 and G2b. Amplification of G1b/2a did not work out. Due to the failure of the restriction digest strategy, we further analyzed the backbone pSB6A1-AraC-lacZ, which we decided to discard and instead, use pSB6A1 and BBa_J04450, which is already available in the parts registry. </p>
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                                   <h1>Week 13</h1>
                                   <h1>Week 13</h1>
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                                   <p style="font-size:10pt; text-align:justify; position:relative; margin-left:6%;">At the beginning of the week, we could verify that the Gibson Assembly for Tripeptide I was indeed positive, however, the other Gibson Assemblies did not work properly. Instead of picking new colonies, we decided to optimize the Gibson recipe instead, as backbone religations were the most common problem. With these improved protocols, we used Gibson Assembly for the Dipeptide, Tripeptide II and Tetrapeptide I, later that week, Tetrapeptide II followed.</p>
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                                   <p style="font-size:10pt; text-align:justify; position:relative; margin-left:6%;">In order to realize the new strategy using the already existing backbone from the parts registry pSB6A1-lacZ-mRFP, we designed two new primers for the backbone amplification and created a map of pHM03.
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The plasmid for the backbone was obtained from the registry, transformed in E.coli and minipreped. The Gibson fragment of the backbone was successfully amplified, together with Gibson fragments DelH G0, G1 and G2b. </p>
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                                   <h1>Week 14</h1>
                                   <h1>Week 14</h1>
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                                   <p style="font-size:10pt; text-align:justify; position:relative; margin-left:6%;">In week 14, we've tested numerous colonies from last week's Gibson assembly 28-07 using DelH G0 as well as G1/2a and 2b by screening-PCR. None of the analyzed colonies carried a correct plasmid. We therefore performed another Gibson assembly 01-08 and again screened numerous clones. We found few possibly correct ones that will be further analyzed next week.
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Additionally, the new D. acidovorans strain SPH1, whose sequence is available in GenBank, was ordered. We will have to amplify all fragments from this strain again as soon as it arrives. </p>
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                                   <h1>Week 15</h1>
                                   <h1>Week 15</h1>
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                                   <p style="font-size:10pt; text-align:justify; position:relative; margin-left:6%;">We further tested colonies from last weeks Gibson assemblies using DelH G0 as well as G1/2a and 2b. Unfortunately, they were all negative in the screening-PCR. Therefore, we amplified our Gibson fragments again and performed more Gibson assemblies. Yet again, there were no positive colonies in the colony-PCR. </p>
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                                   <h1>Week 16</h1>
                                   <h1>Week 16</h1>
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                                   <p style="font-size:10pt; text-align:justify; position:relative; margin-left:6%;">We further characterized the DelH plasmid created using Gibson assembly. Unfortunately, none of the screened colonies was for sure positive. Selection of red colonies was not clear, and PCR screened colonies were all negative.
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In order to avoid high background during screening of the colonies, we decided to run two different strategies. For first approach pHM04, we will amplify the backbone without mRFP, avoiding backbone reassembly due to ribosome binding site homology. In the second strategy pHM05, we will additionally introduce a tetracycline resistance, to ensure integration of the insert.
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In addition to the primers for the new strategies, we designed a new screening primer at the end of DelH. </p>
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                                   <h1>Week 17</h1>
                                   <h1>Week 17</h1>
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                                   <p style="font-size:10pt; text-align:justify; position:relative; margin-left:6%;">After performing different Gibson assemblies and screening always resulted in negative clones, the possible explanation could be a religation of the backbone fragment pSB6A1 allowing E.coli to survive and express mRFP. This week, our aim is to design a new construct without mRFP, so that we can exclude the red colonies from the screening. Therefore, we are going to use a new reverse primer for the backbone including only the terminator of the mRFP, but not the mRFP. The primers for the backbone are HM11 & HM17. The construct is named pHM04 and further explained in week 16. </p>
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                                   <h1>Week 18</h1>
                                   <h1>Week 18</h1>
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                                   <p style="font-size:10pt; text-align:justify; position:relative; margin-left:6%;">Because the constructing pHM04 did not result in any positive clone (see experiments week 17), we will follow the idea to introduce a tetracycline resistance to the ampicillin backbone as additional selection marker for successful assembly. So we have to screen only colonies, which are white (because of the exclusion of mRFP) and grow on plates containing tetracycline. Furthermore, we decided to amplify DelH in various fragments to increase Gibson assembly efficiency. Therefore, we also ordered new screening primers. The primers were designed as shown in the following table. </p>
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                                   <h1>Week 19</h1>
                                   <h1>Week 19</h1>
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                                   <p style="font-size:10pt; text-align:justify; position:relative; margin-left:6%;">After a successful electroporation, we screen numerous clones by colony-PCR and test restriction digest (PvuI-HF). Positive clones are send for sequencing. The sequencing tells us if the DelH Gibson assembly actually worked. Therefore, we send the midipreped plasmids with the reverse DN07 primer or VF2, covering the start sequence of DelH and the end of the pSB6A1 backbone without mRFP (pHM04).
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The screening-PCR showed different positive colonies. After performing the restriction digest, some colonies were discarded and the rest was sent in for sequencing. We did not find any positive clone without a truncating mutation at the beginning of DelH (in the primer region). </p>
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                                   <h1>Week 20</h1>
                                   <h1>Week 20</h1>
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                                   <p style="font-size:10pt; text-align:justify; position:relative; margin-left:6%;">Again, the screening-PCRs resulted in many positive colonies. After the restriction digest, many colonies were kicked out. The remaining minipreped colonies were sent in for sequencing. Yet, we did not identify any positive clone which had no mutation at the beginning of DelH (in the primer region). </p>
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                                   <h1>Week 21</h1>
                                   <h1>Week 21</h1>
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                                   <p style="font-size:10pt; text-align:justify; position:relative; margin-left:6%;">Frustratingly, none of the analyzed clones showed a correct sequence. Probably, DelH by itself is toxic for E.coli and thus, only aberrant clones survive. We suspect the low quality of primers as reason for high number of mutations. Therefore, we will order HPLC purified primers.
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Additionally, we tried to eliminate the mutations in DelH clones I 6b and 15 by mutagenesis. Herefore, primers were ordered. </p>
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                                   <h1>Week 22</h1>
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                                   <p style="font-size:10pt; text-align:justify; position:relative; margin-left:6%;">So far, we failed to find a single correct DelH clone. We suspect the separate DelH module to be toxic for the E. coli. Therefore, they select for mutated plasmids. In order to reduce the selection pressure, we used E. coli BL21 DE3, known for increased expression of the lac repressor. This strategy also did not result in any correct clone. One reason might be, that the E. coli BL21 DE3 we obtained actually are BL21 DE3 pLys, which are unfortunately Chloramphenicol resistant due to additional plasmid and thus not useful for screening and amplification of DelH construct.
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We got new and correct E. coli BL21 DE3 as well as NEB turbo, which significantly overexpress the lac repressor. We however found their lacZ-controlled expression to be very leaky, in contrast to E. coli BL21 DE3.
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Since the Gibson assembly of DelH using the HPLC purified primers also exclusively resulted in mutated clones, we developed two new cloning strategies to avoid expression of DelH. The first strategy uses a weak promoter and RBS, the second introduces DelH in a ccdB helper construct. Last but not least, we continued working on the mutagenesis approach to eliminate the mutation in clone C5. It was sent for sequencing... </p>
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Revision as of 07:46, 2 October 2013

Del H. This bitchy 18 kbp fragment.

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Methods:

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