Parts Construction

      For this project, a total of 13 parts were created, 11 basic parts and 2 composite parts. IDT and Twist Bioscience synthesized the basic parts, and the composite parts were assembled by restriction digestion and ligation in our lab. Each part is compatible with the BioBrick RFC[10] standard. Genes for the TAG lipid production pathway and the IAA pathway were codon optimized for C. minutissima and E. coli, respectively, and a 6xHis tag was added to the end of each coding sequence for convenient protein isolation. 

      However, due to the codon optimization process, many genes had high complexity and could not be chemically synthesized. To solve this problem, sequences were manually modified to maintain the same codons and as much codon optimization as possible while reducing repeat sequences. 

      Some genes, such as DGAT and GPAT, were too complex to synthesize. These genes were synthesized in two parts based on the naturally existing PvuII site near the middle of the gene. This ensured the parts could be synthesized and that our lab could ligate the parts together without having to purchase new materials. 

Escherichia coli

     The genes that we wanted to tranform into E. coli were the iaam and ami1 genes which code for the protein, tryptophan 2-monooxygenase (iaaM) and amidase 1 (ami1) respectively. We used promoters, RBS, and terminators found in the iGem 2022 DNA kit plate. The two that we got the highest concentration of DNA from our midi preps were pSB1C3 plasmids with promoter J23100 (BBa_J364007) and J23151 (BBa_I20270). These were later used to ligate our genes of interest and transform into electrocompetent E. coli cells. 

     pSB1C3 + iaam + J23151 Plasmid Construction 

     pSB1C3 +ami1 + J23100 Plasmid 

     Construction 

     The parts were ligated as shown above, and transformed into electrocompetent Dh5-a E. coli cells. They were then plated on Luria Broth agar plates with chloramphenicol added to it. The colonies were grown in the incubator overnight.

     We picked a colony from each plate and the culture out overnight in our shaker to perform midi preps the next day. We also did a colony PCR using colonies from the plate in order to verify the presence of our ligated product. The readings on the midi prep and the gel from the colony PCR were good so we were certain that we should proceed to perform a protein extraction and western blot to detect any protein expression.

     ami1 + J23151 in pSB1C3 midi prep results:

     iaam + J23100 in pSB1C3 midi prep results:

     Colony PCR on iaam + J23100 and ami1 + J23151 in pSB1C3

     The bands were in the base pair range that we were expecting to see so we went ahead with our protein extraction. However, the procedure was relatively new to us, so human error prevented us from attaining strong promising results during our protein purification.

Chlorella minutissima 

     We chose two plasmids that we had used in our earlier iGem projects, PBI121 and DinoIII plasmids and performed a gel extraction to gain. The homologous arms present in our DinoIII plasmid match our algae, C. minutissima, this makes it ideal for expression in our algae. The ligated LPAAT, DGAT, and GPAT parts will be inserted into both plasmids and transformed into the algae.