Part:BBa_K2945000
Enzyme that converts fatty acids into alkanes.
This gene encodes a fatty acid photodecarboxylase enzyme which converts fatty acids into C1-shortened alkanes and carbon dioxide as a byproduct. This enzyme requires photo-activation by light approximately 450 nm in wavelength in order to be catalytically active. Read more about it from the following two papers doi: 10.1126/science.aan6349 and doi: 10.1002/anie.201807119 .
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)
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 238
Illegal NgoMIV site found at 343
Illegal NgoMIV site found at 1469 - 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI site found at 404
Illegal BsaI site found at 1810
Illegal BsaI.rc site found at 607
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