Figure 1. Conformations of cytochrome b559 in PSII of different species ⁷

PsbE encodes the alpha subunit of cytochrome b559 in photosystem II. It forms a heterodimer with the beta subunit to form cytochrome b559 7. Cytochrome b559 is crucial for the assembly and functionality of photosystem II, mutation in either of the subunits causes the inability of the cell to assemble photosystem II 8. It is thought that cytochrome b559 assists in preventing photoinhibition by accepting excess electrons from electron donors to prevent the formation of reactive oxygen species that will damage the cell 7. The expression of the alpha and beta subunits of cytochrome b559 are controlled by the psbEFLJ operon in tandem repeats whose copy number is controlled by the conditions the cells are grown in whether that be photoheterotrophic conditions or photoautotrophic conditions.

We sought to improve the electricity generation of our cyanobacterial strains and to do so, overexpression of both subunits in cytochrome b559 would allow for more stable assembly of PSII and increase the efficiency of electricity generation. This would also mean that the expression of cytochrome b559 would be more independent of the environment controlled psbEFLJ operon. Furthermore, the anti-photonhibitory properties of cytochrome b559 make it useful for the creation of a biophotovoltaic cell, where the intensities of light can reach up to 2000 umol photons m-2 s-1 9.

Works alongside psbE. PsbF is the beta subunit of cytochrome b559.

In the obligately fermentative bacterium Zymomonas mobilis, the regeneration of NAD+ depends upon two enzymes, pyruvate decarboxylase and alcohol dehydrogenase1. Z. mobilis is the only known obligately fermentative prokaryote that utilizes an Entner-Doudoroff pathway for glycolysis and, like Saccharomyces cerevisiae, produces ethanol and carbon dioxide as dominant fermentation products. Pdc encodes pyruvate decarboxylase which catalyzes the decarboxylation of pyruvate to produce acetaldehyde. In turn, adhB encodes alcohol dehydrogenase II which catalyzes the final step of ethanol fermentation by reducing acetaldehyde to ethanol, using NADH as a cofactor. Introducing adhB and pdc genes in our cyanobacteria would allow it to produce ethanol as a fermentation product and it would be secreted out of the cell into the environment. The use of adhB and pdc for the production of ethanol in cyanobacteria was inspired by the University of Uppsala iGEM 2009 Team2. These parts have been codon optimized for PCC 6803, 7942, and 7002.

To characterize functionality of parts and see if the codon optimization had affected enzyme production in E. coli, we tested BBa_K5052930/BBa_K5052931 in E. coli BL21 and analyzed using Schiff’s reagent. Schiff’s reagent reacts with aldehydes, primary, and secondary alcohols to form a compound that appears as magenta. We used this property of Schiff’s reagent to a) test for the presence of ethanol and b) quantify the amount of ethanol produced when compared to a standard curve.

E. coli BL21 was transformed with BBa_K5052930/BBa_K5052931 and grown at 37C in LB-kan in the dark. Transformants were also plated onto LB-agar-kan plates and left to grow at 37C overnight in the dark. The next day, ethanol was extracted via hydrogel distillation. Liquid cultures were pelleted and supernatant was extracted. 1” x 1” Spenco 2nd Skin Squares hydrogels were soaked in supernatant. 1” x 1” Spenco 2nd Skin Squares hydrogels were laid over cultures grown on solid plates. Before distilling, hydrogels were removed from supernatant and from cultures grown on solid media and were heat treated at 80C to remove any potential cells still remaining on the hydrogel. Hydrogel was then placed on watch glass and covered with 50mL beaker collection vessel and allowed to distill at 80C for 5 min. Distillate collected from collection vessel was moved to 1.5mL microcentrifuge tube. This process for collecting distillate was repeated several times until 100uL of distillate was collected.

This distillate was then tested for ethanol by adding Schiff’s reagent to 10% v/v. Tubes were agitated by inverting and allowed to sit for 1 minute. Observed color change was quantified on ImageJ and compared against a standard curve of 10% Schiff’s added to 10%, 20%, 30%, 50%, and 70% ethanol after incubation for 1 minute.

The IPTG-inducible promoter cLac145 was discovered by Markley et al. 2015 who had sought to design IPTG inducible promoters by modifying existing promoters in cyanobacteria. Pcpc is a strong native promoter in Synechocystis sp. PCC 6803 and by modifying the native Pcpt sequence and replacing sequences flanking the -35 and -10 with lac operator sequences, a new promoter was developed and named PcptOO. By adjusting the core -35 and -10 sequences and the distance between the two operators, Markley et al built a library of cLac promoters each characterized to have slightly different behaviors. It was found that as the PcptOO was mutated to share more homology with the E. coli promoter Ptrc, the responsiveness to IPTG induction increased. cLac145 was made by replacing the Pcpt core of PcptOO with Ptrc and removing the front operator sequence. The result was a strong IPTG inducible promoter with wide dynamic range in cyanobacteria. cLac145 also has the advantage of sharing less homology with native Pcpc present in the Synechocystis sp. PCC 6803 genome which reduces the risk for aberrant homologous recombination in those sites.

To test the functionality of cLac145, this promoter was used in BBa_K5052900 and controlled expression of EYFP. Synechocystis sp. PCC 6803 was transformed with BBa_K5052900 and grown in liquid cultures at 30C with 185 umol photons m^-2 s^-1 shaking at 200rpm in BG11 medium. Cultures were grown for 2 days to an O.D.₇₃₀ of 0.3 prior to testing. cLac145 induction was tested at 250uM, 500uM, 1mM, and 2mM IPTG measuring fluorescence at ex. 488nm and em. 517nm every hour for 8 hours.

The fluorescence of EYFP remains the same regardless of induction concentration. This either suggests a failure in the functionality of cLac145 or that the induction concentration is too low. The concentration of IPTG used for induction should be increased to see expression. This could also be due to the population losing the plasmid containing BBa_K5052900 resulting very little EYFP produced and therefore detected.

Functionality of cLac145 was also tested in E. coli DH5a. DH5a was transformed with BBa_K5052900 which is EYFP controlled by cLac145. Transformants were grown at 37C in LB-kan shaking at 150rpm overnight to an O.D.₆₀₀ of 1.7. Cultures were diluted back to O.D.₆₀₀ of 0.2 before loading 100uL into 96-well plates in triplicates. Like with Synechocystis, cLac145 induction was tested at 250uM, 500uM, 1mM, and 2mM IPTG. The 96-well plate was then scanned on BioTek plate reader at ex. 488nm and em. 517nm for 8 hours at 1 hour increments.

We find that cLac145 is quite responsive in E. coli. This makes sense as the core sequence is derived from E. coli promoter Ptrc. However, it does appear that uninduced DH5a + BBa_K5052900 exhibits higher fluorescence than when induced. Between the samples that were induced, 250uM and 500uM IPTG exhibited similar rates of EYFP production. 1mM and 2mM IPTG showed more expression of EYFP indicating that in DH5a, ideal inducing concentration is 1mM to 2mM IPTG.

To test functionality of PompC in cyanobacteria, we transformed Synechocystis sp. PCC 6803 with composite part BBa_K5052902 which has EYFP expression controlled by PompC. Transformants and WT Synechocystis were grown at 30C under 185 umol photons m^-2 s^-1 shaking at 200rpm until O.D.730 was 0.4. Cultures were diluted back to O.D.₇₃₀ was 0.2 before loading 100uL into 96-well plates in triplicates. 96-well plate was then scanned on BioTek plate reader at ex. 488nm and em. 517nm for 8 hours at 1 hour increments.

The results indicate that PompC doesn’t appear to be responsive in Synechocystis. This is interesting as there is literature indicating that this part is functional in Synechocystis ¹¹.

BBa_K5052102 was also tested for functionality in E. coli DH5a. DH5a was transformed with composite part BBa_K5052902 which has EYFP expression controlled by PompC. Transformants and WT DH5a were grown at 37C shaking at 150rpm until O.D.600 was 1.7. Cultures were diluted back to O.D.600 of 0.2 before 100uL into 96-well plates in triplicates. 96-well plate was then scanned on BioTek plate reader at ex. 488nm and em. 517nm for 8 hours at 1 hour increments.

Our results show activity of PompC in E. coli DH5a. PompC continues to express protein until its peak at around 5 hours in the dark. This promoter proves useful when expressing proteins in cyanobacteria, whose metabolic process are light-dependent. In the absence of light, proteins in an alternative metabolic process can be expressed and allow for unique functionality.

The results indicate that PompC doesn’t appear to be responsive in Synechocystis. This is interesting as there is literature indicating that this part is functional in Synechocystis¹⁰.

BBa_K5052102 was also tested for functionality in E. coli DH5a. DH5a was transformed with composite part BBa_K5052902 which has EYFP expression controlled by PompC. Transformants and WT DH5a were grown at 37C shaking at 150rpm until O.D.₆₀₀ was 1.7. Cultures were diluted back to O.D.₆₀₀ of 0.2 before 100uL into 96-well plates in triplicates. 96-well plate was then scanned on BioTek plate reader at ex. 488nm and em. 517nm for 8 hours at 1 hour increments.

Pcpc560 is a modified promoter from Synechocystis sp. PCC 6803 known as PcpcB which is a strong constitutive promoter controlling the expression of cpcB 1. This result has also been shown to be the case in E coli. The strength of Pcpc comes from its fourteen transcription factor binding sites, as well as two core promoter sequences: P1 and P2 ². The reason Pcpc560 contains two promoters is unclear but it nonetheless contributes to its strength.

To test functionality of Pcpc560 in cyanobacteria, we transformed Synechocystis sp. PCC 6803 with composite part BBa_K5052901 which has EYFP expression controlled by Pcpc560. Transformants and WT Synechocystis were grown at 30C under 185 umol photons m-2 s-1 shaking at 200rpm until O.D.730 was 0.4. Cultures were diluted back to O.D.730 was 0.2 before loading 100uL into 96-well plates in triplicates. A 96-well plate was then scanned on BioTek plate reader at ex. 488nm and em. 517nm for 8 hours at 1 hour increments.

Our results show that Pcpc560 induced expression of EYFP is consistently more than double that of our default control, with the slope constantly increasing. Cyanobacterial metabolic processes are light-dependent, so this promoter proves useful when expressing proteins in the absence of light. Distinct functionality is expressed from these alternative metabolic processes.

We wanted to test Pcpc560 activity in strains other than cyanobacteria to see if it exhibits activity similarly to that of the strain in cyanobacteria. To do this, we transformed E. coli DH5a with BBa_K5052901. Transformants and WT DH5a were grown at 37C shaking at 150rpm until O.D.600 was 1.7. Cultures were diluted back to O.D.600 of 0.2 before 100uL into 96-well plates in triplicates. 96-well plate was then scanned on BioTek plate reader at ex. 488nm and em. 517nm for 8 hours at 1 hour increments.

Our results show activity of Pcpc560 in E. coli DH5a. Pcpc560 continues to express protein until its peak at around 5 hours in the dark. The slope levels off around 5 hours, but then continues increasing around the 7 hour mark after being induced by darkness. This promoter proves useful when expressing proteins in a cyanobacteria chassis, which has light-dependent metabolic processes. In the absence of light, unique functionality is expressed from proteins in an alternative metabolic process.

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EYFP (GenBankID: AAO48599.1) is a fluorescent protein expression codon optimized for cyanobacteria. However, it also works very well in E. coli, thus making it the perfect fluorescent protein for our purposes.

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