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Characterization

Proteorhodopsin

We successfully constructed the part for proteorhodopsin, using PCA assembly based on the information from the paper Light-powering Escherichia coli with proteorhodopsin, as can be verified by our sequencing data in the parts registry. We then conducted an ATP assay on our proteorhodopsin strain to determine if it effectively produced a proton gradient to generate more ATP. Unfortunately our results were inconclusive. In this experiment, we expected to see increased ATP generation when the promoter was induced and light was available for the proteorhodopsin pump. We used cyanide (CN) to inhibit the electron transport chain, and covered tubes in foil to imitate darkness. A 60 Watt incandescent light bulb was used for excitation. aTc was used to bind to the R0040's tetR repressor. Luminescence correlates to ATP concentration.

In the J123106 strain, we saw an unexpected slight increase in luminescence when there was no light source, meaning the proteorhodopsin did not yield increased ATP. The cells treated with CN did not produce markedly higher levels of ATP, suggesting that a 1 mM concentration of cyanide may have been insufficient in shutting down the electron transport chain.

Similarly in the R0040 strain, when aTc was bound to the promoter repressor, we saw only a slight increase in luminescence corresponding to ATP yield. When the repressor was not bound (no aTc was present) the levels of ATP were about the same as when the repressor was bound, signifying the promoter may be a little "leaky".

When we sequenced the proteorhodopsin strain, we found that we had the sequence we expected. However, there are many reasons that our proteorhodopsin did not behave as we expected in the characterization assay. Missing from our part is the ribosome binding site; this could cause lack of expression of the gene. Another issue may be our protocol for reading luminescence. We plan to add a ribosome binding site and research our plate reader protocol for further characterization of the part.

Bacterial Animation

For the results of the bacterial animation project, we have successfully created two constructs using an mCherry plasmid obtained from the Murray Lab at Caltech that we have submitted to the Parts Registry as BioBricks: mCherry-LVA in pSB1C3 and mCherry-AAV in pSB1C3. In parallel we inserted mCherry into R0040. The sequencing data showed all constructs have been built successfully. We ran a RFP assay on our parts in R0040 (with a tetR promoter) and found that our constructs behaved exactly as expected. We ran a characterization assay measuring the fluorescence of R0040, mCherry-LVA, mCherry-AAV, and mCherry (no degradation tag) both with and without aTc, which binds to the tetR repressor. As we hypothesized, the untagged cells produced over 50 times more fluorescent protein than those with degradation tags. The mCherry-LVA/AAV still had more fluorescence than R0040, showing that the protein was produced successfully and the degradation tags worked. Also as we predicted, the LVA had less fluorescence than the AAV, demonstrating that LVA degrades more quickly than AAV.

The picture below shows the same data from the previous graph without the untagged.