Basic Parts

Functional Proteins

cscB

CscB is a cell membrane protein that plays an integral role in transporting sucrose across the membrane as it is a sucrose-proton symporter. It is compatible in many strains of S. elongatus, including 2973, 6803 and 7942 [1]. S. elongatus is unique in that its cell membrane proton gradient is generated by maintaining a surplus of protons inside the cell [2]. Sucrose can then be exported from the cell using the proton gradient. Our codon-optimized BioBrick is 1260 bp long. We made a point mutation at the 383rd base to eliminate an illegal PstI cut site.

EYFP

Currently, BBa_E0030 is a commonly used EYFP BioBrick. We codon-optimized the BioBrick for use in photosynthetic cyanobacteria.

Promoters

PidiA

PidiA is a ferrous-ion repressible promoter native to cyanobacteria. The source of the PidiA DNA is a close relative to our chassis (S. elongatus PCC 7942). The promoter features a palindromic repeat which is suggested to be activated by the binding of idiB, a gene immediately downstream of the idiA gene, in a ferrous deficient environment [3].

Our BioBrick has the 77bp full minimal idiA promoter sequence, plus an upstream AT-rich region [3]. We did not include downstream base pairs due to ambiguity in the true start codon [3].

PcpcB

cpcB codes for phycocyanin, a phycobiliprotein complex, and works to harvest light energy before transmitting it to the chlorophylls. The promoter is a strong promoter [4] but also demonstrates expression patterns following circadian oscillations [5].

PcpcB1A1 features two promoter sequences within the promoter, both of which were included in the BioBrick. For this promoter, derived from Synechococcus elongatus PCC 7942, we kept about 100 bp of upstream and downstream regions, the latter of which was cropped immediately before the ATG start codon. Studies have found that Pcpc can also be used in E. coli.

Pcpc-560

Pcpc-560 is a unique form of the Pcpc promoter native to Synechocystis sp. 6803. It includes the two cpc promoters (as described for Pcpc) and 14 Transcription Factor Binding Sites, allowing it to be an incredibly strong promoter [4] . For the BioBrick, we kept the full 560 bp of Pcpc-560, and did not include an upstream region. The full cpc-560 promoter ends immediately before the ATG start codon in its native form.

PpsbA2

PpsbA2 is a light-inducible promoter native to cyanobacteria [6]. Specifically, it comes from a strain (Synechococcus elongatus PCC 7942) closely related to our chassis organism [7]. The promoter is activated by high light intensity or blue light, and deactivated by low light intensity or red light [8]. The promoter contains a conserved AT-box motif just upstream of the ribosome binding site that serves as the binding site for light-activated proteins [9].

For the Biobrick, we kept most of the original promoter, including a crucial upstream element and the core promoter sequence. Our final Biobrick is 157 bp in length.

References

1. Qiao C, Duan Y, Zhang M, et al. Effects of Reduced and Enhanced Glycogen Pools on Salt-Induced Sucrose Production in a Sucrose-Secreting Strain of Synechococcus elongatus PCC 7942. Appl Environ Microbiol. 2018;84(2): e02023-17. doi: 10.1128/AEM.02023-17.

2. McEwen JT, Machado IMP, Connor MR, et al. Engineering Synechococcus elongatus PCC 7942 for Continuous Growth under Diurnal Conditions. Appl Environ Microbiol. 2013;79(5): 1668-1675. doi: 10.1128/AEM.03326-12.

3. Michel K, Pistorius EK, Golden SS. Unusual Regulatory Elements for Iron Deficiency Induction of the idiA Gene of Synechococcus elongatus PCC 7942. J Bacteriol. 2001;183(17): 5015-5024. doi: 10.1128/JB.183.17.5015-5024.2001.

4. Zhou J, Zhang H, Meng H, et al. Discovery of a super-strong promoter enables efficient production of heterologous proteins in cyanobacteria. Scientific Reports. 2014;4(4500): 1-6. doi: 10.1038/srep04500.

5. Sawaki H, Sugiyama T, Omata T. Promoters of the Phycocyanin Gene Clusters of the Cyanobacterium Synechococcus sp. Strain PCC 7942. Plant Cell Physiol. 1998;39(7): 756-761.

6. Englund E, Liang F, Lindberg P. Evaluation of promoters and ribosome binding sites for biotechnological applications in the unicellular cyanobacterium Synechocystis sp. PCC 6803. Scientific Reports. 2016;6(36640): 1-12. doi: 10.1038/srep36640.

7. Koksharova, OA, Klint J, Rasmussen U. The first protein map of Synechococcus sp. Strain PCC 7942. Microbiology. 2006;75: 664-672. doi: 10.1134/S0026261706060087.

8. Kulkarni RD, Golden SS. Adaptation to High Light Intensity in Synechococcus sp. Strain PCC 7942: Regulation of Three psbA Genes and Two Forms of the D1 Protein. J. Bacteriol. 1994;176(4): 959-965. doi: 10.1128/jb.176.4.959-965.1994.

9. Agrawal GK, Kato H, Asayama M, et al. An AU-bx motif upstream of the SD sequence of light-dependent psbA transcripts confers mRNA instability in darkness in cyanobacteria. Nucleic Acids Research. 2001;29(9): 1835-1843. doi: 10.1093/nar/29.9.1835.

Basic Parts

Functional Proteins

Promoters

References

2018 Stony Brook iGEM

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