Testing the Efficiency of the PAR Promoter in Chlamydomonas reinhardtii
The team USP_UNIFESP-Brazil 2016 first introduced the PAR promoter to iGEM and the team iGEM17_NU_Kazakhstan applied improvements to the part. The PAR promoter is highly important for the iGEM community working with Chlamydomonas reinhardtii since it is a very efficient and faithful promoter. This is why we wanted to further characterize this part. Therefore, we tested the efficiency of the phytobrick BBa_K2516001, the PAR Promoter in C. reinhardtii. It contains the promoters of the proteins HSP70 and RBCS2. Both promoters were fused and as a further improvement the high efficiency separator sequence was introduced. It keeps both binding sides on the same height and therefore improve the efficiency by a factor of 2.6. The RBCS2 promoter is the promoter region for the small subunit gene 2 of RUBISCO and therefore is constitutively expressed. The fusion of the promoters almost completely prevents the heat shock reaction that occurs in vivo with the HSP70A promoter.
It is important to keep improving the iGEM part collection. Therefore, we wanted to test the improvements with the usage in C. reinhardtii. We ligated a Level 2 construct composed of the L1 construct containing spectinomycin resistance with the PSAD promoter and terminator to select positive transformants. To test the PAR promoter the construct contained the coding sequence of the MUT-PETase with the PAR-promoter, a HA-tag to detect the protein and the RPL23 terminator on position two. On position three is the coding sequence of the MHETase with the PAR-promoter, a HA-tag to purify the protein and the RPL23 terminator.
Expression of the enzymes MUT-PETase and MHETase in Chlamydomonas reinhardtii (a) L2A MoClo construct contain aadA selection cassette, PAR-promoter, HA-tagged MUT-PETase and MHETase, RPL23-terminator. (b) UVM4 cultures transformed with construct L2A were inoculated in TAP at 25°C. Samples of eleven different cultures were taken from shake flasks after 3 days. Lysed cells of each sample were loaded onto the gel and proteins were separated via SDS-PAGE. Proteins were blotted onto a membrane and detected by the anti-HA antibody. Molecular weight marker is indicated as MW. Black arrow represents MHETase, white arrow indicates MUT-PETase. The expression via PAR promoter of both MUT-PETase and MHETase is visible in the colonies 18, 22 and 27. The PAR promoter works efficiently in combination with the enzymes MUT-PETase and MHETase. HA-tagged ribosomal chloroplastic 50S protein L5 (RPL5) was used as a positive control.
In summary, we achieved really high expression levels using the PAR promoter. This might be partially because we used the rubisco introns in our coding sequence. As shown in a paper by schroda, the PAR promoter in combination with rubisco introns can lead to extraordinarily high expression levels1,2. To conclude, we can recommend the PAR promoter to anyone working with chlamy. It is the gold standard for a reason and we never felt like we needed to use a different promoter.
[1] Schroda, M., Beck, C. F., & Vallon, O. (2002). Sequence elements within an HSP70 promoter counteract transcriptional transgene silencing in Chlamydomonas. The Plant Journal, 31(4), 445-455.
[2] Lumbreras, V., Stevens, D. R., & Purton, S. (1998). Efficient foreign gene expression in Chlamydomonas reinhardtii mediated by an endogenous intron. The Plant Journal, 14(4), 441-447.
