Coding sequences

We started with the pSB1C3_alsS_ilvC_ilvD_kivD construct which is the part BBa_K1465302. Therefore we used the CDS of the NCTU team Formosa 2011/2012 BioBricks alsS (BBa_K539627), ilvC (BBa_K539621), ilvD (BBa_K539626) and kivD (BBa_K539742). We used the parts of the Parts Kit to combine them by Gibson Assembly. For this we amplified the various CDS and combined them with a RBS (BBa_B0034) by a PCR.
In the beginning it did not worked with pSB1C3 so we used pSB1K3.
When we had pSB1K3_alsS_ilvC_ilvD_kivD, we wanted to reclone it in pSB1C3 and combine it with a promoter. This we wanted to do by BioBrick Assembly. Thereby we identified an illegal restriction side in the end of alsS because of the combination with the following RBS. This we removed by PCR amplification and Gibson with new primer (rv_ilvC_alsS-new, fw_alsS_ilvC-new). In this approach we were able to amplify pSB1C3 as backbone, so no recloning was necessary.

Additionally we wanted to combine our part BBa_K1465302 with the adhA from Lactococcus lactis. This alcoholdehydrogenase was identified as the best enzyme for the last step in the 2-keto-acid pathway (Atsumi2008, Atsumi2010). This pathway is responsible for the isobutanol production.
We wanted to add the adhA to pSB1C3_alsS_ilvC_ilvD_kivD by BioBricck Assembly, but adhA was not available as BioBrick, so we designed it. How we did this you can read in the Results of adhA. We did a BioBrick Sufffix Insertion and the result was the pSB1C3_alsS_ilvC_ilvD_kivD_adhA construct which is the part BBa_K1465303.

Constructs with promoter

To characterize our BioBricks, we wanted to add promoter to them. We chose the ptac promoter (BBa_K731500) for characterizations during cultivations and the stronger T7 promoter (BBa_I719005) for SDS pages.
We performed different BioBrick assemblies, because in the beginning it was not working.
We tried BioBrick Suffix Assembly where the ptac was cut out of the gel and BioBrick Prefix Assembly where we cut the alsS_ilvC_ilvD_kivD part out of the gel. In the end, the suffix version worked out and we created the BioBrick device pSB1C3_ptac_alsS_ilvC_ilvD_kivD (BBa_ K1465306)
The same BioBrick assemblies we performed with the ptac promoter and the alsS_ilvC_ilvD_kivD_adhA part, and the BioBrick Suffix Assembly worked out again. We created the BioBrick device pSB1C3_ptac_alsS_ilvC_ilvD_kivD_adhA (BBa_ K1465307)
For the combination with the T7 promoter we were only performing successful BioBrick Suffix Assembly for both constructs.

pSB1A2_T7_alsS_ilvC_ilvD_kivD

Samples of E. coli KRX with our construct pSB1A2_T7_alsS_ilvC_ilvD_kivD were taken like explained in the cell lysis for a SDS-PAGE Protocol. Protein expression was induced with rhamnose when the culture reached a OD₆₀₀ of 0,8. The first sample was taken right before the induction. Additionally we took samples two, four, 21 and 23 hours later. With these samples, we made a SDS Page. Figure x shows the picture of this SDS Page.

Figure x: SDS page from pSB1A2_T7_alsS_ilvC_ilvD_kivD.
The mass of the overexpressed proteins is 60.78 Da (AlsS), 54.07 Da (IlvC), 65.53 Da (IlvD) and 60.95 Da (KivD)

Several bands seem to increase in their size over the time of sampling. One band at a mass of ~ 68 Da shows a significant difference between the sample of sampling right before the induction and 23 hours later. This could be an evidence of the overexpression of protein IlvD (65.53 Da). As the proteins AlsS (60.78 Da) and KivD (60.95 Da) have almost the same mass you will not see an difference between the band of the proteins in the SDS Page. Right under the band at a mass of ~ 68 Da is a additional band at a mass of ~ 60 Da visible which size increases over the time of sampling, too. This could be an indication for the two proteins AlsS and KivD. Furthermore there is a over time increasing band at a mass of ~ 53 Da apparent. This could be possible the overexpressed protein IlvC (54.07 Da).
These observations fit to our expectations of possible results of this experiment, because all proteins seem to be overexpressed.

pSB1A2_T7_alsS_ilvC_ilvD_kivD_adhA

We took samples of E. coli KRX with our construct pSB1A2_T7_alsS_ilvC_ilvD_kivD_adhA like explained in the cell lysis for a SDS-PAGE Protocol. The inductionof the protein expression with rhamnose happend when a OD₆₀₀ of 0.8 was reached. Right before the induction the first sample was taken and additional samples Protein expression was induced with rhamnose when the culture reached a OD₆₀₀ of 0,8. The first sample was two, four, 21 and 23 hours later. These samples were used for a SDS Page. In figure x you can find the picture of this SDS Page.

Figure x: SDS page from pSB1A2_T7_alsS_ilvC_ilvD_kivD.
The mass of the overexpressed proteins is 60.78 Da (AlsS), 54.07 Da (IlvC), 65.53 Da (IlvD), 60.95 Da (KivD) and 35.78 Da (AdhA)

In the picture of the SDS Page several bands look increasing in their size over the time of sampling. Again bands of approximately the mass of the four proteins AlsS (60.78 Da), IlvC (54.07 Da), IlvD (65.53 Da)and KivD (60.95 Da) seem to be present. The band at a mass of ~ 68 Da could be an evidence of the overexpression of protein IlvD, while right under it the band at a mass of ~ 60 Da could be an indication for the two proteins AlsS (60.78 Da) and KivD (60.95 Da). In addition the over time increasing band at a mass of ~ 53 Da is apparent and could be possible the overexpressed protein IlvC (54.07 Da). Furthermore the size of the band at a mass of ~ 38 Da increases over time and is comparable to the potential band of the AdhA in the SDS Page of pSB1A2_T7_adhA. This band could be the overexpressed protein AdhA (35.78 Da).
As in the SDS Page of pSB1A2_T7_alsS_ilvC_ilvD_kivD all proteins seem to be overexpressed and present. This experiment measured up to our expectations.