Coding sequences

We started with the pSB1C3_alsS_ilvC_ilvD_kivD construct which can be found in the parts registry as 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 distribution to combine them by Gibson Assembly. For this purpose we amplified the various CDS and combined them with the RBS (BBa_B0034) by PCR.
In the beginning it did not work 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. We wanted to do this by BioBrick Assembly. While doing this we identified an illegal restriction site in the end of alsS because of the combination with the following RBS. The restriction site was removed by PCR amplification and Gibson assembly 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 described 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 BioBrick Assembly, but adhA was not available as a BioBrick. So we decided to design it as a new BioBrick (BBa_K1465301). The approach can be found in the Results section 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 different 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 1 shows the picture of this SDS Page.

Figure 1: SDS page from pSB1A2_T7_alsS_ilvC_ilvD_kivD.
The mass of the overexpressed proteins is 62,004 Da (AlsS), 54,069 Da (IlvC), 65,532 Da (IlvD) and 60,947 Da (KivD)

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 induction of the protein expression with rhamnose happened 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 samples were taken two, four, 21 and 23 hours later. These samples were used for a SDS Page. In Figure 2 you can find the picture of this SDS Page.

Figure 2: SDS page from pSB1A2_T7_alsS_ilvC_ilvD_kivD_adhA.
The mass of the overexpressed proteins is 62,004 Da (AlsS), 54,069 Da (IlvC), 65,532 Da (IlvD), 60,947 Da (KivD) and 35,776 Da (AdhA)