Team:Heidelberg/Delftibactin/DelH

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                        <h1><span style="font-size:180%;color:#FFCC00;">Del H.</span><span class="text-muted" style="font-size:120%"> This nasty 18 kb fragment.</span></h1>
                        <p style="font-size:14px">Facing the challenge to clone 18 kbp of genomic DNA from <i>D. acidovorans</i>.</p>
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                                      <h1>Week 1</h1>

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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 <i>D. acidovorans</i> 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 retrieved 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 2</h1>
                                  <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 generated by restriction digest. New BioBricks to obtain the AraC promotor (I13453 and K206000) were chosen. 
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                                  <h1>Week 3</h1>
                                  <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 primers DN07:Screen_DelH_rev and DN13:Screen_DelH_fw to check for correct ligation of the DelH fragment F1. 
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                                  <h1>Week 4</h1>
                                  <p style="font-size:10pt; text-align:justify; position:relative; margin-left:6%;">The elongation of the pSB6A1-AraC-lacZ backbone turned out to be difficult. So in week 4, we performed different restriction digests both with our final backbone pSB6A1-AraC-lacZ and with the former construct pSB1C3-AraC-lacZ to verify the identity of the backbone. Besides, the amplification of the DelH fragment F1 was planned: it will be amplified in 2 subfragments - fragment F1a and fragment F1b (both 5 kb in size). </p>
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                                  <h1>Week 5</h1>
                                  <p style="font-size:10pt; text-align:justify; position:relative; margin-left:6%;">This week, we started over with the assembly of the backbone pSB6A1-AraC-lacZ: digesting AraC, lacZ and PSB6A1 based on the previously amplified fragments. The amplification of DelH was continued and we succesfully amplified all three fragments and gel-extracted them. </p>
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                                  <h1>Week 6</h1>
                                  <p style="font-size:10pt; text-align:justify; position:relative; margin-left:6%;">Since all DelH-fragments required for the final construct as well as the backbone were assembled in week 5, we tried to assemble 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. The screening via colony-PCR was negative, so none of the transformed <i>E.coli</i> received the correct plasmid. </p>
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                                  <h1>Week 7</h1>
                                  <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 that amplification of fragments F1a and F1b was not reproducible. Therefor, we designed new primers for DelH, which will allow 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 yielded best results and will be used in the next experiments. </p>
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                                  <h1>Week 8</h1>
                                  <p style="font-size:10pt; text-align:justify; position:relative; margin-left:6%;">After improving the amplification of the first subfragment of DelH (fragment 1a with the primer DN11), we used a higher concentration in the ligation assembling the pHM01 plasmid. The transformation of <i>E.coli</i> DH10ß cells was performed with electroporation. Four colonies were positive in the screening-PCR, but did not express lacZ (no blue color). To qualitatively determine DelH expression, we plan to conduct SDS-PAGEs of cell lysate derived from DelH transformed cells. Also, we will induce the transformed bacteria with higher concentrations of arabinose and X-Gal.
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                                      <h1>Week 9</h1>

                                      <p style="font-size:10pt; text-align:justify; position:relative; margin-left:6%;"> SDS-PAGE of cell lysate derived from DelH-transformed with subsequent coomassie staining did not yield a band at the expected protein size of ~600 kDa. The result was confirmed on DNA level, as the restriction digest of the DNA prepped from colonies of the transformed cells did not show the expected pattern. To perform a new assembly of plasmid pHM01, the fragments of DelH and the backbone had to be reamplified. The amplifications of the DelH fragments were successful (confirmed by gelelectrophoreses), but the backbone caused difficulties and we were not able to amplify it. 
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                                  <h1>Week 10</h1>
                                  <p style="font-size:10pt; text-align:justify; position:relative; margin-left:6%;">After unsuccessful assembly attempts of pHM01, we tried to verify the previously amplified fragments. Two difficulties were encountered:  On the one hand, the concentrations of the fragments were too low and could not be detected by gelelectrophoresis; on the other hand, a re-amplification of the fragments with the appropriate primers was not successful. For the DelH 1b fragment, the PCR products of the amplification from genomic DNA were slightly larger than the reamplifications. 
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                                  <h1>Week 11</h1>
                                  <p style="font-size:10pt; text-align:justify; position:relative; margin-left:6%;">Based on this week's experiments and <i>in silico</i> analysis of 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 an alternative method, developed a strategy and designed primers accordingly. 
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                                  <h1>Week 12</h1>
                                  <p style="font-size:10pt; text-align:justify; position:relative; margin-left:6%;">We started amplifying the Gibson fragments using different PCR approaches. Since we were not able to amplify G1 by PCR, we decided to divide G1 into the subfragments G1a and G1b, for which we had already designed and ordered primers. The amplification of fragment G2 worked well and yielded sufficient DNA amounts for subsequent steps. Alternatively, we also produced the entire DelH G0 as one fragment, as well as further subfragments 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. Instead, we choose to use pSB6A1 and BBa_J04450, which is already available in the parts registry. </p>
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                                  <h1>Week 13</h1>
                                  <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.
The plasmid for the backbone was obtained from the registry, transformed into <i>E.coli</i> and miniprepped. 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>
                                  <p style="font-size:10pt; text-align:justify; position:relative; margin-left:6%;">In week 14, we screened numerous colonies from last week's Gibson assembly (28-07) for plasmids containing DelH G0, G1/2a and 2b by screening-PCR. None of the analyzed colonies carried the correct plasmid. We performed another Gibson assembly (01-08) and screened numerous clones. We found few possibly correct ones that will be further analyzed next week.
Additionally, the new <i>D. acidovorans</i> strain SPH1, whose sequence is available in GenBank, was ordered. We will amplify all fragments from the genome of <i>D. acidovorans</i> SPH1 as soon as it arrives. </p>
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                                  <h1>Week 15</h1>
                                  <p style="font-size:10pt; text-align:justify; position:relative; margin-left:6%;">We screened colonies from last weeks Gibson assemblies (01-08) for plasmids containing DelH G0 as well as G1/2a and 2b. None of the screening-PCRs yielded the expected DNA bands. Therefore, we amplified the Gibson fragments again and performed further Gibson assemblies. Yet again, we could not detect positive colonies by colony-PCR. </p>
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                                  <h1>Week 16</h1>
                                  <p style="font-size:10pt; text-align:justify; position:relative; margin-left:6%;">We further characterized the DelH plasmid created by Gibson assembly. None of the screened colonies yielded definit positive results. Selection of red colonies was not clear, and PCR screened colonies were all negative.
In order to avoid high background during screening of the colonies, we decided to run two different strategies. First, we will amplify the backbone without mRFP, avoiding backbone reassembly due to ribosome binding site homology (pHM04). In the second strategy (pHM05), we will additionally introduce a tetracycline resistance to select for the insert via antibiotic resistance.
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>
                                  <p style="font-size:10pt; text-align:justify; position:relative; margin-left:6%;">A possible explanation for the failed Gibson assemblies could be a religation of the backbone fragment pSB6A1 allowing <i>E.coli</i> to survive. In this case, transformed bacteria will still express mRFP. This week, our aim is to design a new construct without mRFP (pHM04), by which we will be able to exclude red colonies from the screening. For this strategy, we are going to use a new reverse primer for the backbone still including the terminator of mRFP, but omitting mRFP itself. The primers for the backbone amplification are HM11 & HM17.</p>
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                                  <h1>Week 18</h1>
                                  <p style="font-size:10pt; text-align:justify; position:relative; margin-left:6%;">Since the assembly strategy for pHM04 did not yield positive clones (see experiments week 17), we will follow the idea to introduce a tetracycline resistance to the ampicillin backbone as an additional selection marker for successful assembly. With this approach, positive clones can be easily determined by their white phenotype (exclusion of mRFP) and their ability to 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>
                                  <p style="font-size:10pt; text-align:justify; position:relative; margin-left:6%;">After successful electroporation, we screened numerous clones by colony-PCR and test restriction digest (PvuI-HF). Midiprepped DNA dervied from clones positive for both methods were sent for sequencing. By sequencing of the transition sequence from the end of the pSB6A1 backbone without mRFP (pHM04) to the beginning of DelH, the assembly success of the Gibson assembly can be determined (Primer: reverse DN07 primer or VF2).Sequencing results showed truncating mutation at the beginning of DelH (in the primer region) for all clones. </p>
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                                  <h1>Week 20</h1>
                                  <p style="font-size:10pt; text-align:justify; position:relative; margin-left:6%;">As in week 19, the screening-PCRs showed colonies positive for the DelH contatining plasmid, whereas the restriction digest reveiled many of the screening results to be false positive. The remaining miniprepped colonies were sent in for sequencing. Sequencing results showed again truncating mutation at the beginning of DelH (in the primer region) for all clones. </p>
                                  
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                                  <h1>Week 21</h1>
                                  <p style="font-size:10pt; text-align:justify; position:relative; margin-left:6%;">None of the analyzed clones showed a correct sequence, which lead as to the assumptions following assumptions. First, we suspect DelH to be toxic for <i>E.coli</i>, thus only clones carrying the mutant DelH-plasmid survive. Second, we consider the low quality of the gibson primers as a possible explanation for the high number of mutations in the assemblies. To circumwent the latter problem, we will order HPLC purified primers.
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>
                                  <p style="font-size:10pt; text-align:justify; position:relative; margin-left:6%;">So far, we failed to obtain a single correct DelH clone. We suspect the DelH module to be toxic for <i>E. coli</i> when transformed without the other parts of the Del cluster, thus DelH-transformed cells would select for mutated plasmids. In order to reduce the selection pressure, we used <i>E. coli</i> 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 <i>E. coli</i> BL21 DE3 strain we obtained was actually a BL21 DE3 pLys strain, which itself is already Chloramphenicol resistant, thus not useful for screening and amplification of the DelH construct coded for on a chloramphenicol vector.
We obtained the correct <i>E. coli</i> BL21 DE3 strain as well as NEB <i>E. coli</i> turbo cells, which significantly overexpress the lac repressor. We found the lacZ-controlled expression of the latter to be very leaky when compared to <i>E. coli</i> BL21 DE3.
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 ribosomal binding site, the second introduces DelH in a ccdB helper construct. Lastly, we continued working on the mutagenesis approach to eliminate the mutation in clone C5. It was sent for sequencing... </p>
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                                  <h1>Week 25</h1>
                                  This week we tried to clone DelH into a plasmid with neither promotor nor ribosome binding site (pFS_03). We managed to obtain two clones which did not have the mutations usually observed at the beginning of DelH. This proves that DelH is in fact toxic. In parallel we constructed another plasmid (pFS_04) into which the correct DelH should then be ligated by common restriction enzyme based cloning.
                                  
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