Results

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Results

C. reinhardtii Culture

Our 2023 iGEM project is the first time our lab has attempted to culture C. reinhardtii, and we are excited to have successfully cultured this microalgae. We based our protocol on the 2016 Linkoping University iGEM team, and we found that this protocol worked well for our lab.


Parts Construction

We successfully transformed our genes for PHB expression, ordered from IDT, into E. coli. To begin building our expression part, we successfully transformed CaMV35S (BBa_J428074), mCherry (BBa_J428079), and eukaryotic terminator (BBa_J42808) into E. We verified that our transformation was successful through colony PCR and extracted the plasmid through mini-prep.


Figure 1 Left- Gel results simulated in SnapGene. Lanes 1 contains the eukaryotic terminator. Lanes 2 and 3 contain mCherry. Lanes 4 and 5 contain CaMV35S. Right- Our experimental gel showing colony PCR results is consistent with our simulated gel.


Figure 1

With a large amount of our kit plate parts secured, we began a process of restriction digest and ligation to attach these genes to our IDT-synthesize PHB genes. We designed four expression parts, one for phaA (BBa_K4736009), phaB (BBa_K4736010), phaC (BBa_K4736011), and our reporter gene mCherry (BBa_K4736008).


Figure 1 mCherry composite part for expression


Figure 1

Figure 2 phaA composite part for expression


Figure 2

Figure 3 phaB composite part for expression


Figure 3

Figure 4 phaC composite part for expression


Figure 4

However, after transforming our ligated expression part into E. coli and running colony PCR, our experimental gel does not show the expected results, suggesting that contamination may have occurred. We will begin the restriction digest and ligation process again, and we expect to have a successful transformation and have tested for PHB production by the 2023 iGEM Jamboree.


Figure 5 Gel results simulated in SnapGene. Lanes 2 and 3 contain the phaA expression part. Lanes 4 and 5 contain the phaB expression part. Lanes 6 and 7 contains the phaC expression part. Lanes 8 and 9 contain the mCherry expression part.


Figure 4