General

Our plan was to create multiple composite parts, which included CooA. To be pure the DNA needed to have a A260/280 value between 1,80-2,00 and a A260/230 value between 2,00 and 2,20. These composite parts would have different promoters and output genes, so we can measure carbon monoxide in multiple ways.

For further steps we isolated the DNA. The DNA had to be pure for digestions. To be sure the DNA was pure, requirements were set. Hereby there would be looked at the curve of the line also. If the line wasn’t smooth, the DNA was most likely not pure. A pure sample has a curve like shown in figure 1.

To produce enough DNA of BBa_J04450 (an plasmid containing a functional spacer) we performed multiple mini preps (DNA isolations), which gave us the following results:

Successful isolated DNA could be digested and ligated with another biobrick for testing. Early named biobricks were paired with each other, where BBa_K133071 and BBa_K173003 functioned as vectors and BBa_I134353 and BBa_J23100 as inserts. We used the backbone from the vectors, so we only needed to insert 1 biobrick into another biobrick. To know if the ligation had worked the DNA was transformed into the E.coli strain: NEB10Bèta. Colonies that grow were tested by digesting again. The digested DNA was put on gel, and the length was determined.

CooA production

When we started our project our first order of business was to reliably produce CooA, the carbon monoxide dependent transcription factor. We knew we had to constitutively produce CooA, and had to produce as much as we could to elicit a high enough response, but we also knew that our cells wouldn’t be able to produce it indefinitely. To this end we decided to combine different constitutive promoters (BBa’s J23100, J23105 and J23113), and combine them with different ribosomal binding sites (BBa’s B0030, B0031 and B0032). This could then be combined with CooA (BBa_K352001) and a double terminator (BBa_B0015).

At first we tried to insert our ribosomal binding site into the plasmids with the different promoters, though because the backbones of the promoters were different from the standard registry backbones we had some difficulty with the cloning, so we decided to turn our plan around and tried to insert the promoters into the plasmids containing the different ribosomal binding sites. The next problem we had with our cloning however was the size of the promoters themselves. As they are only around 15 base pairs long, and the ribosomal binding sites are around 35 base pairs long, when combined the total change in plasmid size would only be around 50 to 60 base pairs, which we couldn’t visualize on our electrophoresis gels.

After this conundrum we had decided to follow the standard iGEM 3A assembly method, using BBa_J04450 (pSB1C3, pSB1K3 and pSB1A3 versions of this biobrick) as a quick screening method.

After a few attempts we seemed to have successfully cloned BBa_J23105 with BBa_B0032 and BBa_K352001 with BBa_B0015. This has then been cloned together to make the full CooA production plasmid. Of the resulting colonies we made 10 different colony PCR’s, and put these on gel, of which one seemed to be the expected size.

Sadly when we sequenced the CooA part in pSB1A3, the sequence conformed to the sequence of BBa_J04450, which is about the same length as the expected CooA product from the PCR.