Summary
Aims:
- To characterise the three components of a UV-A sensor system: uirS, uirR, PlsiR.
- To investigate E. coli survival rates in UV-A and sunlight.
Results:
Parts:
- Essential:
• K1725400 (PlsiR)
• K1725410 (UirS)
• K172541#(UirS with RBS)
• K1725420 (UirR)
• K1725421 (UirR with RBS)
- Others:
• K1725401 (PlsiR.I13500)
• K1725402 (PlsiR.E5501)
• K1725422 (J23101.B0032.UirR)
• K1725423 (J23110.B0032.UirR)
• K1725424 (J23114.B0032.UirR)
• K1725425 (J23116.B0032.UirR)
• K1725426 (J23101.B0032.UirR.B0015)
• K1725427 (J23110.B0032.UirR.B0015)
• K1725428 (J23114.B0032.UirR.B0015)
• K1725429 (J23116.B0032.UirR.B0015)
• K1725430 (J23101.B0032.UirR.B0015.PlsiR.I13500)
• K1725431 (J23110.B0032.UirR.B0015.PlsiR.I13500)
• K1725432 (J23114.B0032.UirR.B0015.PlsiR.I13500)
• K1725433 (J23116.B0032.UirR.B0015.PlsiR.I13500)
Overview
In order to prevent cells permanently shifting their metabolism towards one that favours light production, we decided to repress the bioluminescence genes (our optimised lux operon) in the presence of sunlight. The chosen system recognises unidirectional UV-A light and comes from the cyanobacterium synechocystis sp. PCC6803. The system has not yet been characterized by an iGEM team for the Parts Registry. Three components are required to produce a response to UV-A: uirS (UV intensity response Sensor), uirR (UV intensity response Regulator), and PlsiR (Promoter of the light and stress integrating response Regulator) (Song et al., 2011). Expression of a chromophore molecule, phycocyanobillin (PCB), is also required for functional light sensing. Phycocyanobillin is synthesised in a two step pathway from Heme within cyanobacterium. Several biobricks encoding the two phycocyanobillin synthesis pathway enzymes already existed within the Registry of Parts due to utilisation of PCB in other previously characterised synechocystis sp. PCC6803 light sensors such as the ccaS green-light sensor system.
We obtained uirS and uirR from synechocystis genomic DNA via PCR. Our primers were flanked with the BioBrick prefix as well as the medium strength ribosome-binding site B0032, and the biobrick suffix. For uirS we utilised the reverse primer to modify the stop codon from the (suboptimal in E. coli) TAG to TAA.
A G-Block was ordered from IDT of the 535bp genomic region identified in Song et al. between the stop codon of uirR and start of lsiR, which was demonstrated to contain the uirR transcriptional activator binding region for the promoter of lsiR. The G-block was synthesised with the biobrick prefix and suffix attached, and a SpeI site within mutated away by one base (t58a).
The uirR gene and <PlsiR promoter region were both biobrick compatible and were therefore ligated directly into the pSB1C3 plasmid; whereas uirS possessed three BioBrick incompatible restriction sites, preventing ligation into pSB1C3 and submission to the registry.
In order to make uirS biobrick compatible we first had to clone it into a vector which would allow us to alter the incompatible restriction sites by PCR mutagenesis. We chose to use the TOPO® TA Cloning® Kit from Invitrogen (450641) and followed the protocol provided (See protocols). Through a diagnostic digest of 5 miniprepped transformants it was observed that uirS inserted into TOPO vector reversed in all 5 cases. TOPO cloning is a non-directional process, but it was hypothesised at this stage that the combination of the lac promoter and very high copy number resulting from the pUC origin of replication present in pCR 2.1 TOPO vector lead to selection against expression of uirS.
Once successfully cloned into a vector we began the process of PCR mutagenizing the BioBrick incompatible restriction sites in a stepwise manner using the QuikChange II Site-Directed Mutagenesis Kit from Agilent (200523) (See protocols). The Agilent QuikChange Primer Design tool was used to design mutagenic primers to modify a single base in the three illegal restriction sites without altering the amino acid sequence of uirS and attempting to match codon frequency. Although we were modifying uirS in its reversed conformation within TOPO vector, the following sequence labels were numbered in the forward direction from the start codon.
• XbaI site (1): 571-576
o TCTAGA to TCTcGA = a573c
o CTA (Leucine) to CTC (Leucine)
• EcoRI site (2): 1638-1643
o GAATTC to GAATcC = t1641c
o ATT (Isoleucine) to ATC (Isoleucine)
• XbaI site (3): 1790-1795
o TCTAGA to TCTcGA = a1792c
o AGA (Arginine) to CGA (Arginine)
Figure 1: Plasmid map of uirS after ligating into pCR 2.1 TOPO vector reversed. Generated by ApE (A Plasmid Editor).
The final step prior to beginning mutagenesis was performing polyacrylamide gel purification of the primer oligonucleotides and quantifying them with a spectrophotometer (See protocols).
The first illegal site modified using the Quikchange protocol was a1792c, followed by t1641c, then finally a573c. In order to track our samples the initial
uirS.TOPO colonies were numbered 1 to 5, and DNA from colony 1 was used in the mutagenesis of a1792c. Following each mutagenesis several transformant colonies were given letter designations e.g. A-D and checked by diagnostic digest to confirm successful silencing of the incompatible site, then the DNA of one correct colony was chosen to move to the next step.
Interestingly, it transpired that the XbaI site of a573c was protected by Dam methylation;
E. coli Dam-methylase methylates the Adenines of GATC sequences. XbaI sequence is TCTAGA, and at this position in
uirS was flanked by TC, leading to methylation in the centre of the restriction site and inability for XbaI to bind and cut at the location. In practice this would also have meant that
uirS would be biobrick compatible without removing this XbaI site, as there would not be interference with use of XbaI for plasmid assembly, however the Parts Registry would still reject the part as BioBrick incompatible under the RFC10 standard as it does not consider methylation when processing part DNA sequence. Additionally, in order to visualise the silencing of a573c by restriction digest the use of a Dam Methylase knockout (Dam-) strain of
E. coli (NEB: ER2925) was required. Plasmid DNA that is purified by miniprep from ER2925
E. coli lacks Dam methylation and thus the XbaI site can be cut and the fragments visualised. The other option to confirm successful mutagenesis of the site would have been DNA sequencing, but we had a sample of ER2925 available to use.
We used the program
ApE to create plasmid maps and digest predictions, the following figure shows the expected digest results at each stage of our PCR mutagenesis, if the miniprep DNA was cut with EcoRI and XbaI restriction enzymes.
Figure 2: Predictive digest of stepwise removal of biobrick incompatible restriction sites. Generated by ApE: A Plasmid Editor.
Miniprep DNA from each step (
uirS1,
uirS1A,
uirS1AA, and
uirS1AAA) was transformed into the ER2925 Dam- strain, a colony was cultured, miniprepped, and digested with EcoRI-HF and XbaI-HF enzymes. Undigested DNA from each sample was run in lanes 1-4.
Figure 3: Restriction digest. Lanes 1-4 ; Uncut uirS1-1AAA. Lanes 6-9 uirS1-1AAA digested with EcoRI and XbaI. DNA miniprepped from Dam- methylation strain ER2925 so methylation protected XbaI site at position 523 can be checked by digest. 1% agarose gel stained with Ethidium bromide and photographed on a Bio-Rad GelDoc. Figure illustrates the removal of restriction sites in a sequential manner, with the final uirS1AAA sample being biobrick compatible.
Once
uirS was biobrick compatible it was digested from pCR 2.1 TOPO plasmid and ligated into pSB1C3, then further light-sensor plasmid assembly could take place.
Sensor
In synechocystis, the uirS two-component system is involved in a negative phototactic response to unidirectional UV-A light (Song et al., 2011). The proposed mechanism places uirS, a transmembrane protein, as the protein that photosenses UV light. It is suggested that a phosphotransfer from uirS to uirR occurs upon UVA-light excitation of Phycocyanobillin chromophore bound within uirS; uirR is released from the transmembrane protein and locates to a binding region upstream of the lsiR promoter. uirR bound upstream of lsiR is shown to act as a transcriptional activator of lsiR (Song et al., 2011). Figure BLAH is the illustration of the uirS mechanism taken from Song et al.
Figure 4: Figure taken from Song et al., 2011. Illustrating the proposed mechanism of the uirS two-component UV-A photosensory system in synechocystis sp. PCC6803.
The
uirS system possesses structural and functional homologies with the ccaS green-light photosensor system, also from
synechocystis sp. PCC6803, which has been characterised by several previous iGEM teams (Edinburgh 2010, Wash U St Louis 2014). Both sensors are two-component systems where a bound Phycocyanobillin chromophore detects light within a transmembrane sensor protein (CcaS,
uirS), leading to phosphorylation of a DNA binding regulatory protein (CcaR,
uirR), which binds upstream of a promoter region (cpcG2,
PlsiR) inducing transcriptional activation. A 2014 paper by Schmid et al. “Refactoring and Optimization of Light-Switchable Escherichia coli Two-Component Systems”, detailed the optimisation of the
ccaS green-light sensor system. We hoped that the investigations into optimised plasmid assembly for
ccaS could be applied to our
uirS UV-A light sensor system due to their homologies.
The paper reports that for the
ccaS sensor, the most effective assembly is a two plasmid arrangement; where the sensor and PCB synthesis genes are placed on a low copy number p15A origin of replication plasmid, while the response protein, associated promoter, and reporter genes are placed on a high copy number ColE1 ori plasmid. Figure BLAH is taken from Schmid et al and shows their optimised green light sensor assembly.
We have confirmed through the use of a laser scanner that PlsiR is not active when UirS and UirR are absent. PlsiR was ligated to GFP with two ribosome binding sites of different strength and no fluorescence was observed (the parts we used for this experiment were K1725401 and K1725402) (Chart 1). Moreover, cells that possess UirR but lack UirS also did not show levels of fluorescence above the expected for E. coli. Therefore, UirR is not sufficient to drive the activation of PlsiR.
Chart 1. Relative Fluorescence (Compared to Last Taken Measurement of Constitutively Expressed GFP Control) over Absorbance in DH5α cells. DH5α cells containing the PlsiR promoter with GFP fluoresce no more than the original laboratory strain or cells that have GFP without a promoter.
Survivability
Introduction
As our genetic circuit contain a UV-A sensor, we decided to test how Uv-A exposure affected different strain of E. coli in order to decide on a chassis. It is well known that UVB can be lethal to E. coli, but some strains might be able to withstand the dose of UV-A required to activate out UVA sensor (50µM/m2/sec).
Initial aims
We decided to use a survivability assay to choose a chassis strain. TOP10, DH5α, DS941 and MG1655 strains were our possibilities. UV light causes mutations, so the strain must be able to repair it's DNA. We predicted that the recA- mutants (TOP10 and DH5α) would be adversely affected by UVA exposure, whereas, DS941 is a recF mutant, so may be less adversely affected by UVA exposure. MG1655 (wild-type for recombination) was used a control strain.
Method
1ml 10x serial dilutions were made up to 10-6 from a 5ml overnight of each strain. 10µl spots of each dilution were then spotted onto LB agar plates.
These plates were then exposed to 50µM/m2/sec of UVA at room temperature in illumination cabinets. Time points were then taken by removing plates from the illumination cabinet.
After illumination, plates were incubated at 37°C, under the assumption that every single viable cell will form a colony. The length of incubation is irrelevant, provided that every cell is given enough time to form a visible colony. This also forms the basis of our counting system, where a colony is assumed to have come from a single cell. We also make the assumption that cell division at the temperature and time we were running the experiment was negligible/nonexistent.
After incubation, the colonies on each spot/dilution were counted. The number of colonies from the lowest visible dilution (some dilutions formed a lawn of growth) were then multiplied by the dilution factor to approximate how many cells would be in 10µl of undiluted culture.
Results
As shown in Figure 7, TOP10 and DH5α cell counts drop dramatically after 20 minutes. DS941 and MG1655 show some variation, but do not appear to be as adversely affected as the recA- mutants. Therefore DS941 was chosen as our chasis.
Figure 7: Estimated cell count per 10ul of a 5ml overnight in lb broth over time of exposure to 50µM/m2/sec to UVA
Conclusion
Read More!
