Part:BBa_K5052101
Pcpc560
About
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 remains unclear, but it is hypothesized to contributes to its strength.
Testing Functionality
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 wild type (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. A 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 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 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 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.
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.
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 3.
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) Gupta, J. K.; Srivastava, S. The Effect of Promoter and RBS Combination on the Growth and Glycogen Productivity of Sodium-Dependent Bicarbonate Transporter (SbtA) Overexpressing Synechococcus Sp. PCC 7002 Cells. Front Microbiol 2021, 12, 607411. doi.org/10.3389/fmicb.2021.607411.
(2) Zhou, J.; Zhang, H.; Meng, H.; Zhu, Y.; Bao, G.; Zhang, Y.; Li, Y.; Ma, Y. Discovery of a Super-Strong Promoter Enables Efficient Production of Heterologous Proteins in Cyanobacteria. Sci Rep 2014, 4, 4500. doi.org/10.1038/srep04500.
(3) 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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