Team:Heidelberg/Delftibactin/MMCoA

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                        <h1><span style="font-size:140%;color:#FFCC00;">Methylmalonyl-CoA Pathway.</span><span class="text-muted" style="font-family:Arial, sans-serif; font-size:90%"> Making the whole thing work.</span></h1>
                        
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                                      <h1>Week 6</h1>

                                      <p style="font-size:10pt; text-align:justify; position:relative; margin-left:6%;">In this week the first experiments for the genomic integration of the methyl-malonyl-CoA synthetase were set up. First of all the plasmids were transformed into TOP10 grown and purified. The first attempts on the amplification of the fragment didn't work at all and we ended up with a specific product of the wrong size.</p>
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                                      <h1>Week 7</h1>

                                      <p style="font-size:10pt; text-align:justify; position:relative; margin-left:6%;">After a validation of the template plasmid via restriction digest the PCR reaction was optimised. Unfortunately none of the cells transformed with the PCR product using electroporation could be positively screened, no matter how the PCR fragment was amplified.</p>
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                                      <h1>Week 8</h1>

                                      <p style="font-size:10pt; text-align:justify; position:relative; margin-left:6%;">As the repetition of the colony PCR under different conditions was still negative, new PCR products were prepared and pooled. To eliminate possible errors fresh competent cells were prepared.</p>
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                                      <h1>Week 9</h1>

                                      <p style="font-size:10pt; text-align:justify; position:relative; margin-left:6%;">After doing colony PCR again and getting inconclusive results, it was decided that the genomic integration failed using this experimental setup. Thus we started from scratch using a new primer pair and finally it turned out that the polymerase was causing the problems. By the end of the week BAP1-pKD46 could be transformed.</p>
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                                      <h1>Week 10</h1>

                                      <p style="font-size:10pt; text-align:justify; position:relative; margin-left:6%;">Even after running several colony PCRs there were no positive clones to be found. Thus the pKD46 was digested for validation. On the gel a miniprep from the PCR-transformed cells was included. The identity of the pKD46 was fine and the miniprep didn't contain any DNA, as expected.</p>
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                                  <h1>Week 11</h1>
                                  <p style="font-size:10pt; text-align:justify; position:relative; margin-left:6%;">In this week the whole procedure was repeated again from fragment amplification to extensive colony PCR. This week several different colony types were encountered and one that grew only after 3 days actually gave a band at 1.2 kb, but it turned out to be unspecific.</p>
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                                  <h1>Week 12</h1>
                                  <p style="font-size:10pt; text-align:justify; position:relative; margin-left:6%;">Several possible error sources such as unselective plates were investigated, but all turned out to be fine. The colony PCR was then repeated using yet another primer pair, but it still only gave unspecific or no bands.</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 check whether the cell line used was actually the right one primers against sfp were used and cell from other subgroups were included. It turned out that something really weird was going onwith the cells, since sfp could not be detected. Thus the entirely new strategy of generating a plasmid instead of doing genomic integration was initialised.</p>
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                                  <h1>Week 14</h1>
                                  <p style="font-size:10pt; text-align:justify; position:relative; margin-left:6%;">To be really sure about the sfp a new plasmid preparation was made and cells were tranformed again. In parallel the pLF03 was also prepared and sent for sequencing. It turned out, that there is a deletion in the sequence and thus the primers can't bind properly.</p>
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                                  <h1>Week 15</h1>
                                  <p style="font-size:10pt; text-align:justify; position:relative; margin-left:6%;">A final try on integrating the methyl-malonyl-CoA pathway was made. At the same time new plasmids carrying the pathway arrived and had to be validated first via PCR and restriction digestion. Several PCR reaction were run and also the first Gibson assembly and transformation was done for both plasmids. The positive colonies were sent for sequencing by the end of the week.</p>
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                                  <h1>Week 16</h1>
                                  <p style="font-size:10pt; text-align:justify; position:relative; margin-left:6%;">After finally giving up on the genomic integration we focused on the plasmid strategy, where the sequencing results of the two plasmids arrived. As they were already positive glycerol stocks were prepared.</p>
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                                  <h1>Week 17</h1>
                                  <p style="font-size:10pt; text-align:justify; position:relative; margin-left:6%;">In order to check for sfp expression pMM64, carrying an indigoidine synthetase was cotransformed, but didn't give any blue colonies. Thus the plasmid backbone had to be changed to allow for cotransformation with our own indigoidine construct. One of the new plasmids was sent for sequencing.</p>
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                                  <h1>Week 18</h1>
                                  <p style="font-size:10pt; text-align:justify; position:relative; margin-left:6%;">The sequencing results of pIK1.3 revealed a point mutation in the permeability device. All the other plasmids sent for sequencing also carried some kind of mutation that introduced a stop codon within the permeability device, which is thus non-functional. Moreover there were still no blue colonies even though we now used our own construct.</p>
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                                  <h1>Week 19</h1>
                                  <p style="font-size:10pt; text-align:justify; position:relative; margin-left:6%;">After doing some research in 2007's Cambridge team wiki it was found, that the permeability device is probably toxic to the cells and can thus only be succesfully cloned behind a very weak promotor.</p>
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                                  <h1>Week 20</h1>
                                  <p style="font-size:10pt; text-align:justify; position:relative; margin-left:6%;">This week we grew the first blue colonies with an updated version of the indigoidine construct. We also put the permeability device behind a very weak promotor and prepared new plasmids for sequencing.</p>
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                                  <h1>Week 21</h1>
                                  <p style="font-size:10pt; text-align:justify; position:relative; margin-left:6%;">When the sequencing results were analysed there were no mutations in the permeability device. Now the cloning of the plasmids for the parts submission started and by the end of the week we sent them for sequening.</p>
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                                  <h1>Week 22</h1>
                                  <p style="font-size:10pt; text-align:justify; position:relative; margin-left:6%;">Unfortunately we noticed that we accidentally introduced an additional restriction site and could thus not submit the part to the registry. We already removed the restriction site and validated it by restriction digestion. Anyway we are going to try different restriction enzymes and send it for sequencing after wiki freeze.</p>
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