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Team:Valencia/Parts
From 2012.igem.org
Submitted Parts
psbAI promoter
We have placed the strong psbAI promoter of Synechococcus elongatus PCC7942 into the Standard Biobrick, pSB1C3, in order to create an assembly standard for this promoter and allow other users to assemble it together with other parts to create longer and more complex parts suitable to work in cyanobacteria and photosynthetic organisms. You can find more information about standards here.
Sequence
1 ggactagagg ctggatttag cgtcttctaa tccagtgtag acagtagttt tggctccgtt
61 gagcactgta gccttgggcg atcgctctaa acattacata aattcacaaa gttttcgtta
121 cataaaaata gtgtctactt agctaaaaat taagggtttt ttacaccttt ttgacagtta
181 atctcctagc ctaaaaagca agagttttta actaagactc ttgcccttta caacctcaag
241 atcgat
Description
The psbAI gene of Synechococcus elongatus PCC 7942 is one of three psbA genes involved in the coding of a fundamental PSII reaction center protein: D1. This family of genes is regulated distinctly in response to changes in the light environment resulting in an interchange of two different forms of the D1 protein. The expression of psbAI is downregulated under high intensity light, while the other two members are induced. (Nair et al. 2001).Function
Cyanobacteria, as well as algae and higher plants, carry out oxygenic photosynthesis, which requires multiprotein complexes that driven by solar energy produce reducing power (NADPH) and chemical energy (ATP). In this system water is the source of electrons in reducing C02 to various organic compounds. The PSII is involved in the water oxidation reaction and the release of oxygen and its core is composed of two critical proteins D1 and D2 (figure 1), which coordinate the cofactors of light-driven charge separation (Andersson and Styring, 1991). Due to the strong oxidative chemistry of the PSII, the D1 protein is subjected to constant photooxidation stress and therefore requires regular replacement to guarantee a steady-state level of D1 protein under different environmental conditions. Under low light growth, the rate of replenishment is 5h, while under intense illumination, the protein is replaced every 20 minutes (Tyystjärvi et al. 1994). In cyanobacteria the three psbA genes that encode the D1 protein are under strict regulation to guarantee the proper functioning of the PSII. In Synechococcus elongatus PCC7942 this three genes encode two distinct D1 protein isoforms: D1:1 being encoded by psbAI and D1:2 by psbAII and psbAIII (Golden et al. 1986).
Regulation
One strategy of the psbA regulation is to replace the D1 protein under unstressed conditions with a different form when high-light intensity is detected. The other strategy is upon stress, to increase the turn-over of the same D1 protein produced under basic growth conditions (Mulo et al. 2009) (figure 2). The three psbA genes are regulated at both transcriptional and posttranscriptional levels. Under low light conditions (125 μE m-2 s-1) over 80% of the transcripts are from psbAI, however after high light conditions (750μE m-2 s-1) psbAII and psbAIII message levels increase (Bustos et al. 1991).
Regulation of the psbAI gene and functional elements of the psbAI promoter
One of the most crucial determinants of gene expression in cyanobacteria is the initiation of transcription, where several sigma factors are involved in promoter recognition (Mulo et al. 2009). The psbAI promoter has characteristic -35 spaced elements from the E. coli σ70 promoter, but has an atypical -10bp element TCTCCT (Golden et al. 1986) (figure 3), which entails that this promoter doesn't work in E. coli (Schaefer and Golden, 1989) making it difficult to characterize it properly. The smallest psbAI functional promoter region comprises nucleotides -54 to +1, and one or more proteins bind specifically to the psbAI upstream region stimulating, rather than inactivating the transcription (+1 to + 43) (Nair et al. 2001), unlike typical σ70 promoters. A segment of approximately 20bp of the consensus -35 element has been shown to be implicated in both, promoter activation per se and light-responsive expression, this region is characterized by being AT-rich (Nair et al. 2001).
Behaviour
Some studies show that psbAI transcript is actively destabilized when shift to high light (Kulkarni et al. 1992) (figure 4, 5 and 6), but prolonged exposure of S. elongatus PCC 7942 cells to high light leads to an increased accumulation of all psbA transcripts, including psbAI (Kulkarni and Golden, 1994). This is an electronic flow independent response implying a response to light rather than to redox changes (Tsinoremas et al. 1996). Extensive assays suggest that the psbAI promoter in S. elongatus is among the strongest in this organism (Andersson et al. 2000).
Using this part in S. elongatus
If you would like to use this part in Synechococcus elongatus PCC7942 you should be aware that the transformation of S. elongatus is based on homologous recombination between two sites on the chromosome (neutral sites) that have been developed as cloning locus. Ectopic sequences can be homologous recombinant without any apparent aberrant phenotype (Clerico et al. 2007).
Thus, to use this part you should cloned it within the S. elongatus neutral sites sequences to move it into the cyanobacterial chromosome.
When transforming, the selective marker and the part of interest flanked with the neutral site sequences are inserted into the neutral site of S. elongatus chromosome and the backbone is lost.
To read more about techniques concerning site-directed mutagenesis in Cyanobacteria, please take a look at this paper:
Clerico, Eugenia M., Ditty, Jayna L., Golden, S.S. (2007) Specialized Techniques for Site-Directed Mutagenesis in Cyanobacteria. Methods in Molecular Biology. 362:153–172.
Promoter source
We sincerely want to thank Prof. Susan Golden for providing us with a psbAI:luxABCDE fusion construct ready to use in Synechococcus elongatus PCC7942 and the appropriate protocols and papers to achieve our goal. Furthermore, her research on this promoter helped us to prepare a standard characterization report for the registry.
The synthesis of the psbAI promoter for our Biobrick was carried out by Genscript. In the protocols page you will find the ligation protocol we followed to assemble our part into psb1C3.
References
Andersson, B. & Styring, S. (1991) Photosystem II: molecular organization, function, and acclimation. Curr. Top. Bioenerg . 16:1–81.
Andersson, C. A., Tsinoremas, N. F., Shelton, J., Lebedeva, N. V., Yarrow, J., Min, H. & Golden, S. S. (2000) Application of bioluminescence to the study of circadian rhythms in cyanobacteria. Methods Enzymol. 305:527–542.
Bustos, S. A & Golden, S. S. (1991) Expression of the psbDII gene in Synechococcus sp. strain PCC 7942 requires sequences downstream of the transcription start site. J. Bacteriology, 173:7525–33.
Clerico, Eugenia M., Ditty, Jayna L., Golden, S.S. (2007) Specialized Techniques for Site-Directed Mutagenesis in Cyanobacteria. Methods in Molecular Biology. 362:153–172.
Golden, S. S., Brusslan, J. & Haselkorn, R. (1986) Expression of a family of psbA genes encoding a photosystem II polypeptide in the cyanobacterium Anacystis nidulans R2. EMBO J., 5:2789–98.
Golden, S. S. (1995) Light-Responsive Gene Expression in Cyanobacteria. J. Bacteriology, 177:1651–1654.
Kulkarni, R. D. & Golden, S. S. (1995) Form II of D1 is important during transition from standard to high light intensity in Synechococcus sp. strain PCC 7942. Photosyn. Res. 46:435–443.
Kulkarni, R. D., Schaefer, M. R. & Golden, S. S. (1992) Transcriptional and posttranscriptional components of psbA response to high light intensity in Synechococcus sp. strain PCC 7942. J. Bacteriol. 174:3775–3781.
Mackey, S.R., Ditty, M.J., Clerico, M. E. & Golden, S. S. (2007) Detection of rhythmic bioluminescence from luciferase reporters in cyanobacteria. Methods in Molecular Biology, 362:115–29.
Mulo, P., Sakurai, I. & Aro, E. M. (2012) Strategies for psbA gene expression in cyanobacteria, green algae and higher plants: from transcription to PSII repair. Biochimica et biophysica acta, 1817:247–57.
Mulo, P., Sicora, C. & Aro, E.M. (2009) Cyanobacterial psbA gene family: optimization of oxygenic photosynthesis. Cellular and molecular life sciences : CMLS, 66:3697–710.
Nair, U., Thomas, C. & Golden, S. S. (2001) Functional Elements of the Strong psbAI Promoter of Synechococcus elongatus PCC 7942. J. Bacteriology, 183:1740–1747.
Schaefer, M.R. & Golden, S. S. (1989) Light availability influences the ratio of two forms of D1 un cyanobacterial thylakoids. J. Biol. Chem, 264:7412–7417.
Schmitz, O., Tsinoremas N. F., Anandan, S. & Golden, S. S. (1999) General effect of photosynthetic electron transport inhibitors on translation precludes their use for investigating regulation of D1 biosynthesis in Synechococcus sp. strain PCC 7942. Photosyn. Res. 62:261–271.
Tsinoremas, N. F., Schaefer, M- R & Godel, S. S. (1994) Blue and Red Light Reversibly Control psbA Expression in the Cyanobacterium Synechococcus sp. Strain PCC7942. J. Biol. Chem, 269:16143–16147.
Tsinoremas, N. F., Ishiura, M., Kondo, T., Andersson, C. R., Tanaka, K., Takahashi, C. H., Johnson, C. H. & Goldem, S. S. (1996) A sigma factor that modifies the circadian expression of a subset of genes in cyanobacteria. EMBO J. 15:2488–2495.
Tsinoremas, N. F., Kawakami, A. & Christopher, D. A. (1999) High-fluence blue light stimulates transcription from a higher plant chloroplast psbA promoter expressed in a cyanobacterium Synechococcus (sp. strain PCC7942). Plant cell Phys., 40:448–52.
Tyystjärvi, T., Aro, E. M., Jansson, C., Mäenpää , P. (1994) Changes of amino acid sequence in PEST-like area and QEEET motif affect degradation rate of D1 polypeptide in photosystem II. Plant Mol. Biol. 25:517–526.
