Part:BBa_K5052102
PompC
About
PompC is a dark-inducible promoter from E. coli. It has been shown to also work in cyanobacteria, such as Synechocystis sp. PCC 6803 1. We chose this promoter to induce reactions that will co-occur with the light-independent stages of cyanobacteria metabolism.
Testing Functionality
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 30 °C under 185 µmol photons m-2 s-1 shaking at 200 rpm until O.D.730 was 0.4. Cultures were diluted back to O.D.730 was 0.2 before loading 100 µL into 96-well plates in triplicates. 96-well plate was then scanned on BioTek plate reader at ex. 488 nm and em. 517 nm 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 1.
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 37 °C shaking at 150 rpm 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. 488 nm and em. 517 nm 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.
Parts Collection
Synthetic biology heavily relies on modularity and the ability for parts to be exchanged and adjusted easily. However, little focus has been placed on modularity within composite parts, and when they are, the focus is usually the DNA coding sequence. To add to the synthetic biology toolbox, our team has developed a collection of parts of promoters that can easily be swapped. This allows for a wider range of flexibility and modularity to express parts under different conditions, where one can start testing functionality of a BioBrick under the context of one promoter before swapping to another to fine-tune a BioBrick’s expression without needing to clone a new part. This expedites the design-build-test-learn cycle by saving time and resources that would have been spent cloning new parts.
The promoter swap system is based on traditional cut-and-paste cloning. Traditional cut-and-paste cloning was an attractive solution to us, as the smallest of recognition sites can be easily added without disturbing the function of the full composite part. Two restriction sites are placed on either end of the promoter, DraIII cut site is placed upstream of the promoter and MluI cut site is in between the ribosome binding site and the start codon of the DNA coding sequence. Promoters can be exchanged via double digest with DraIII and MluI, performing a gel extraction of the vector, and inserting your new promoter of choice.
We have worked to modify three cyanobacterial promoters: cLac145–an IPTG inducible promoter (Part: BBa_K5052100), Pcpc560–a constitutive promoter (Part: BBa_K5052101), and PompC–a dark inducible promoter (Part: BBa_K5052102).
We characterized our promoter swap system and measured success by comparing EYP expression in parts with promoters swapped to their native sequence. We took EYFP controlled by PompC (BBa_K5052902) and cLac145 (BBa_K5052900) and swapped out the promoters. For PompC + EYFP, we cut out PompC with DraIII and MluI to replace it with cLac145 and separately with Pcpc560. For cLac145 + EYFP, we cut out cLac145 with the same pair of restriction enzymes to replace it with PompC.
After performing traditional cut-and-paste to insert the respective promoters, the ligated products were used to transform DH5a E. coli. These transformants were grown on LB-agar-kan plates at 37 °C in the dark overnight. The next day, these plates were taken for fluorescent imaging.
This image was quantified for fluorescence on ImageJ by taking the area of the plate and calculating the intensity of the fluorescence. With this value, we can divide by the background values to find the corrected total cell fluorescence 2.
When comparing swapped promoters to their native sequence, we see that the levels of fluorescence are vastly different. cLac145 + EYFP has a corrected total cell fluorescence of 11.93 compared to after we swapped it with PompC where the corrected total cell fluorescence increased to 16.321. When looking at PompC + EYFP, it shows a corrected total cell fluorescence of 18.875 which decreased greatly to 10.066 when swapped with Pcpc560 and to 11.354 when swapped with cLac145. Furthermore, comparing strength of fluorescence between parts sharing the same promoter show similar results, like between cLac145 + EYFP and PompC insert cLac145 which have a corrected total cell fluorescence of 11.93 and 11.354 respectively.
Our parts collection is only the beginning. Any promoter can be modified to fit our collection by fitting a DraIII cut site upstream and an MluI cut site in between the RBS and DNA coding sequence.
References
(1) Immethun, C. M.; DeLorenzo, D. M.; Focht, C. M.; Gupta, D.; Johnson, C. B.; Moon, T. S. Physical, Chemical, and Metabolic State Sensors Expand the Synthetic Biology Toolbox for Synechocystis Sp. PCC 6803. Biotechnol. Bioeng. 2017, 114 (7), 1561–1569. doi.org/10.1002/bit.26275.
(2) Measuring cell fluorescence using ImageJ — The Open Lab Book v1.0. theolb.readthedocs.io/en/latest/imaging/measuring-cell-fluorescence-using-imagej.html (accessed 2024-10-02).
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
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