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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)
    • K1725411 (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. The forward primer used was flanked with the BioBrick prefix as well as the medium strength ribosome-binding site B0032, with the biobrick suffix flanking the reverse. We also utilised the reverse primer for uirS to modify the stop codon from TAG (suboptimal in E. coli) 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 4 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 5
is taken from Schmid et al and shows their optimised green light sensor assembly.
Figure 5. Optimised ccaS green light sensor plasmid maps, taken from Schmid et al. 2014.


We replicated as closely as possible the two-plasmid assemblies with our uirS system biobricks, creating two composite parts: K1725430 (J23101.B0032.uirR.B0015.PlsiR.GFP) and K1725445 (J23106.B0032.uirS.K322122(ho1 and pcyA)). To emulate copy number, in testing we utilised the pSB1C3 high copy number pMB1 plasmid for the K1725430 response-reporter plasmid, and pSB3K3 p15A low copy for the K1725445 sensor-chromophore plasmid.

As our chassis for UV-A testing we chose the E. coli strain DS941, which exhibited increased survivability under UV illumination when compared to both TOP10 and DH5α in our UVA illumination survival study. DS941 cells carrying K1725430.pSB1C3 plasmid were made chemically competent (Protocols) and transformed with the K1725445.pSB3K3 plasmid. Resulting transformants were cultured, miniprepped, and checked with a diagnostic digest to check presence of both plasmids. Three of the transformants checked appeared correct under diagnostic restriction digest. We then designed an experiment to determine whether sunlight UVA illumination would result in expression of GFP from our assembled uirS two plasmid system.

Six E. coli strains were tested:
  • 3 different DS941 transformants expressing both sensor and response plasmids
  • DS941 with K1725430 Response plasmid alone
  • DS941 with K1725445 Sensor plasmid alone
  • DS941 cells only

Cells were cultured overnight then 1 ml of o/n was added to 20 ml of fresh broth in a 100 ml conical flask, and incubated with shaking @ 37C for 2 hours. Then 10 ml of each cell culture was added to two empty Petri Dishes, one for sunlight illumination and one to be kept in darkness. Sunlight plates were placed on a tray by a window, and darkness plates were wrapped in tinfoil. After 5 hours 1ml of cells from each plate was pippetted into fresh eppendorf tubes, spun down to a pellet in a microfuge, and then resuspended in Phosphate binding solution (PBS: see interlab study protocol for full methodology). 200 l of each cell type was placed in a microwell plate along with blank PBS as a control, and scanned with a Typhoon fluorescence reader (see interlab study protocol for full methodology).

No fluorescence above that exhibited by the cells-only control wells was observed, indicating that our assembly of the uirS two component system did not function. At this point there was not sufficient time remaining prior to the jamboree to do further testing to discern the reason for the failure of the system, however the two reasons we believe to be the most likely cause of failure are:
  • PlsiR promoter from Synechocystis does not function as a promoter in E. coli. It may not be able to recruit the sigma-70 factor required for binding of E. coli RNA-polymerase to initiate transcription
  • The Phycocyanobillin synthesis biobrick used K322122 may be supplied with a mutated promoter, leading to lack of expression of PCB. We have documented this error in sequencing of its promoter on the experience page on the part K322122



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

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Protocols

Bioluminescence

Terminator

Repressors

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