Characterize a Part BBa_J61100: The Strength of this ribosome binding site has previously been shown to be extremely week, but up to this point, it has not been evaluated using the protocol that was designed as a result of the 2018 Interlab Study. UChicago set out to characterize this RBS using the Interlab Protocol. To do this, we attached this promoter RBS construct to BBa_I746916 which is a super folder GFP. We then followed the Protocol for a plate reader experiment using this construct, a blank pSB1C3 vector, and a blank non-iGEM vector.
From the plot below, we can definitely see that there are small amounts of green fluorescent protein (gfp) present in our ligation sample. We have chosen to use fluorescent measurement as a means to measure our ribosome binding site efficiency because the ribosome binding sites connect the promoter with an iGEM gfp. Since there is gfp present, this means that our characterization was successful. Our empty vector control shows small amounts of gfp due to the nature of our promoter BBa_J23119. Our negative control shows similar readings as the background readings on our plate reader, which means that our negative control is successful because there are no gfp present. This serves as a good reference for our sample comparison. The finding that there is no significant difference between our negative control and the gfp part in fluorescence confirms team Delft's measurements that this ribosome binding site is weak(found here). Furthermore, a non-iGEM vector (adapted from Rauch et al, 2019) showed lower fluorescence than did the iGEM vector with and without the gfp gene inserted. This indicates some auto-fluorescence of the iGEM vector which limits the feasibility of the 2018 interlab protocol when characterizing the fluorescence of low efficiency parts.
FASyn Operon: For the FASyn operon, we encountered many errors with gibson assembly and ultimately decided to create individual plasmid constructs for many of the relevant genes in the Fatty acid synthesis pathway. This would enable us to individually troubleshoot any issues with each individual gene and also provided the necessary groundwork for our future directions with this project. Below are the chromatograms showing DNA sequencing results which indicate successful creation of FabD, FabG, and FabF. Note that FabG was not assembled into a plasmid and thereby is only a gene fragment whereas FabD and FabF were assembled into plasmids with promoters and ribosome binding sites.
Fab D Chromatogram
Fab F Chromatogram
Fab G Chromatogram
Alk Operon: To validate the functionality of the alkane part, we tested our part in E coli rather than Synechococus due to the growth time difference. Growth curve experiments demonstrated that cells with the Alk operon transformed grew at a significantly slower rate compared to their control (see exporter operon results below). This suggests that the Alk part is mildly toxic to the cells which corresponds to its predicted effect of producing alkanes. To further confirm this was due to a functionality of the Alk part rather than the protein itself, we performed Gas Chromatography Mass spectrometry to confirm the existence of alkanes. It was clear that cells that were allowed to grow overnight in the presence of white light to provide the blue light needed to activate the enzyme. The gas chromatogram, when compared to control, clearly indicated a difference in composition (Figure 2a), but a search of the NIST Mass Spec Library suggested that this was Triton-X, not the alkanes we originally thought (Figure 2b).
Figure 2a: Comparison of Gas Chromatograms of hexane cell extract from FAP cells (bottom) and negative control (top)
Figure 2b: Mass Spectrum of retention time 6-6.2 min of FAP cells (bottom). Spectrum of Negative Control (top)
It wasn’t until one lab member forgot to clean up samples and left them out for 5 days that different results began to emerge. After 5 days at RT, all of the negative control solutions had turned pink while the supernatants transformed with the Alk Operon remained white as during the initial preps (Figure 3). Once Again, GCMS analysis was run on both samples after hexane extraction. The Chromatograms do not indicate a distinct difference (Figure 4a). However, when Mass Spec plots were generated at the 6.1-6.2 retention time, the composition of those plots was quite different. The sample chromatogram showed characteristic peaks produced by long chain alkanes while the control peaks were more characteristic of the previously observed Triton-X (Figure 4b). Search of the NIST Mass Spec Library top 9 hits further supported these results (Report 1). This demonstrates that our Fatty Acid Photocarboxylase enzyme works in converting fatty acids in the cells to alkanes, thereby allowing for efficient biofuels production.
Figure 3: Photographs of samples that were left out for 5 days at RT. FAP sample (Left) and Negative control (Right)
Figure 4a: Gas Chromatograms of samples after 5 days. FAP (Top two) and negative control Bottom (Bottom two)
Figure 4b: Mass Spectrum of time point 6.1-6.2 of FAP (left) and negative control (Right) critical
Figure 4c: Mass Spectrum of time point 6.0 of Alkane Standard (Sigma Aldrich)
Exporter Operon: The exporter operon was the only plasmid that was properly synthesized and FLAG-tagged for Western Blot. A sequencing chromatogram can be seen below.
We constructed growth curves to test the exporter operon's impact on growth rate both with and without the presence of the Alk Operon. As is shown below, growth with the Exporter (AlkL) operon seems to follow that of the control quite closely. The Alk Operon, however, ends at a lower plateau, which is likely due to alkane toxicity. Growth with both operons plateaus with an even lower optical density. A possible reason for this lower plateau is the presence of two antibiotics (ampicillin and chloramphenicol).
To test the effect of two antibiotics, a secondary growth curve was constructed. This secondary growth curve test compares cells with both Alk and AlkL operons to control cells with empty vectors with both antibiotic resistances. The cells with both operons reach a lower plateau than that of the control cells. This suggests that despite being an importer, AlkL's passivity does allow for alkanes to exit the cells when the intracellular concentration reaches a high enough point. For the Alk+AlkL cells though, this point is reached when the alkane levels within the cell are already too toxic, and growth has slowed.
Furthermore, we performed a Western Blot to validate the expression of our channel protein in E. Coli. As is shown in the image below, we were able to detect a faint band of the proper size - 26 kDa.