Part:BBa_K5052100
cLac145
About
cLac145 was discovered by Markley et al. 2014 who had sought to design IPTG inducible promoters by modifying existing promoters in cyanobacteria1. 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 cyanobacterial. 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.
Testing Functionality
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 30 °C with 185 µmol photons m-2 s-1 shaking at 200 rpm 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 250µM, 500µM, 1mM, and 2mM IPTG measuring fluorescence at ex. 488 nm and em. 517 nm 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 150 rpm overnight to an O.D.600 of 1.7. Cultures were diluted back to O.D.600 of 0.2 before loading 100 µL into 96-well plates in triplicates. Like with Synechocystis, cLac145 induction was tested at 250µM, 500µM, 1mM, and 2mM IPTG. The 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.
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, 250µM 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.
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) Markley, A. L.; Begemann, M. B.; Clarke, R. E.; Gordon, G. C.; Pfleger, B. F. Synthetic Biology Toolbox for Controlling Gene Expression in the Cyanobacterium Synechococcus Sp. Strain PCC 7002. ACS Synth Biol 2015, 4 (5), 595–603. doi.org/10.1021/sb500260k.
(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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