Part:BBa_K754000
S. elongatus PCC7942 psbAI promoter
Our part is the Synechococcus elongatus promoter for gene psbAI. This promoter works on a constitutive way, althought its activity can be enhanced or decreased in the presence or absence of light (in nature it works regulated in a circadian cicle). This part is suitable for building light-induced devices.
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]
Usage and Biology
The psbAI gene of the cyanobacterium Synechococcus elongatus PCC 7942 is one of three psbA genes that encode a critical photosystem II reaction center protein, D1. Regulation of the gene family in response to changes in the light environment is complex, occurs at transcriptional and posttranscriptional levels, and results in an interchange of two different forms of D1 in the membrane. Expression of psbAI is downregulated under high-intensity light (high light) in contrast to induction of the other two family members.
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, 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).
Functional Parameters
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). 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 loci. Ectopic sequences can be homologous recombinant without any apparent aberrant phenotype (Clerico et al. 2007). Thus, to use this part you should clone it within the S. elongatus neutral sites sequences and incorporate 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.
How to use this part
Transforming S. elongatus PCC7942 for promoter characterization:
Our main objective has been to characterize the psbAI promoter (Submitted parts) of Synechococcus elongatus PCC7942 in order to know more about its operation and understand how this promoter would control our final construct ( Designed parts) to have a diel switch of AHL, the signal molecule for our Aliivibrio fischeri population to glow.
To characterize this promoter we had two options: making psbAIp::lacZ fusions and monitored the β-galactosidase activity or making psbAIp::luxABCDE fusions and monitored the bioluminescence produced as a result of the promoter activation. We refused to use a psbAIp::lacZ fusion because some assays report that the psbAI promoter response to light cannot be properly monitored by the β-galactosidase activity (Nair et al. 2001). For this reason we choosed using vector fusions. With this aim, we cloned several fusion plasmids (pAM977 and pAM2195) in Escherichia coli and transformed them into S. elongatus, both wildtype and cscB strain. The fusion vectors were provided by Susan Golden´s lab.
Cloning into E. coli
We successfully cloned all the vectors in DH5α E. coli strains. For E. coli transformation we used this protocol (link). For the selective selection for pAM977 we used ampicillin (1μl/ml) and for pAM2195 Chloramphenicol (0.7μl/ml). The lack of spectrophotometer or other apparatus to measure optical density in our lab impede us to determine the concentration of our vectors.
Transforming Coccus
Once cloned we transformed a wild type strain provided from the Physiology, Genetic and Microbiology departments of University of Alicante and the cscB strain provided from the Wyss Institute in Harvard. As the cscB strain from Harvard was resistant to Cm, we only could transform it with the pAM977 plasmid, which gives Sp resistance. Wildtype was transformed with both plasmids (pAM977 and pAM2195). We used the following protocol to transform Synechococcus:
">Protocol
Extracted from Clerico et al. 2007:
We grew 100ml of cscB and WT in BG-11 liquid medium (see how we grown them and our recipe here) shaking at 250rpm with constant light we used cold light fluorescent tubes. Cells had an optical density at 750nm (OD750) of 0.7, which is reach in 4 to 7 days approximately.
We collected 15mL of the culture and centrifuge it at 6000g for 10 min and discarded the supernatant.
We resuspended collected cells in 10 mL of 10mM NaCl and centrifuge them again at 6000g for 10 min.
After it, we resuspended the pellet in 0.3mL of BG-11M liquid medium.
We added 2μl of pAM2195 and pAM977 after doing a Miniprep of the clonation with a JetQuick kit (link al kit?)
Wrap tubes in aluminum foil to keep out light and incubate at 30oC for 15 to 20h with gentle agitation.
And then, plate the entire 0.3mL cell suspension on BG-11 medium agar (see here the recipe) with the selective antibiotics. We used this concentration: 2μg/ml spectinomycin and 7.5μg/mL of chloramphenicol. After 7 to 10 days of incubation under standard light conditions transformed colonies should appear.
After colonies appearance pick single transformants and grow them on fresh liquid BG-11M agar plate with selective antibiotic (this is done to ensure that all colonies have incorporated the trans gene as S. elongatus has multiple copies of its chromosome).
After 5 to 7 days of growth, cyanobacteria can be used to inoculate a BG-11 liquid culture with the selective antibiotic.
Results
We weren´t able to obtain transformants with the wild type strain of S. elongatus. But we obtained resistant colonies to Sp of the cscB strain transformed with the pAM977 vector (figure x). This was achieved the last week of lab work, so due to the slowly growth rate of S. elongatus, we haven´t have the time to grow the transformants in liquid medium and start with the bioluminescence measures to characterize the promoter. However, we will try to have some results for the Jamboree concerning this part.
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