Results
CLONING
This section describes our efforts to construct pCAM and pTOD, and the reporter plasmids for assaying promoter activity. The construction process consists of two steps: (a) PCR amplification or restriction digestion to obtain insert fragments and linearised vectors, and (b) cloning, involving either Gibson Assembly or conventional digestion-ligation cloning. Constructs were verified either by colony PCR or by restriction digestion.
Constructing pCAM and pTOD
The CamC (1335bp), CamA (1365bp), and CamB (402bp) fragments were PCR amplified using PrimeSTAR Max from the pP450cam, pPdR, and pPdX plasmids provided by Dr. Teruyuki Nagamune. The sequences synthesised by IDT arrived in pIDTSmart plasmids and the TodC1 (1440bp), TodC2-TodA (1011bp), and TodB (1311bp) fragments were PCR amplified using PrimeSTAR Max. The vectors pSEVA2213 (3862bp) and pSEVA4313 (3221bp) were linearised by PCR for multi-fragment Gibson Assembly to construct pCAM and pTOD after digestion failed repeatedly.
With multi-fragment Gibson Assembly failing consistently, we resorted to single-fragment assembly. The CamC, CamA, and CamB fragments and the TodC1, TodC2, TodA, and TodB fragments were fused by PCR to produce CamCAB (2964bp), and TodC1C2AB (3624bp), respectively. This involved PCR amplifying the fragments together in a PrimeSTAR Max reaction for ~15 cycles with long annealing and extension times, after which primers were added to the mix and the reaction was run for an additional ~30 cycles.
We were unfortunately unable to solve the issues that plagued our cloning process and could not assemble pCAM or pTOD in the time available to us, but we have designed revised constructs that would enable us to carry this project forward if we had additional time.
Hypoxic promoters
Pseudomonas putida KT2440 genomic DNA was extracted using the CTAB method and used as a template to PCR amplify the extended ANR-inducible promoter of ccoN1 (399bp) and introduce homology regions. The vector pSEVA2213 (3820bp) was linearised by PCR for promoter insertion.
The plasmid pPccoN1 was constructed using In-Fusion Assembly and verified by colony PCR (399bp amplicon). Glycerol stocks were prepared for storage.
pSEVA2213 (3862bp) and pPccoN1 (4430bp) were digested with XbaI and SalI, and pET28a-sfGFP was digested with XbaI and XhoI to cut out the sfGFP coding sequence (787 bp).
pPccoN1-sfGFP and pPEM7-sfGFP were assembled by ligating the digested vector
and the insert overnight with T4 Ligase. Clones were verified based on the
inherent fluorescence of the colonies for pPEM7-sfGFP. We did not obtain
positive colonies for pPccoN1-sfGFP (4640bp).
pPEM7-sfGFP was linearised by PCR with the inherent EM7 promoter deleted in this process, and the amplicon was digested to create sticky ends for ligation. Annealed oligo cloning was attempted with this vector, but we did not obtain successful clones despite repeated attempts. We suspect that this may be due to inefficient digestion and have designed a replacement set of primers to introduce alternate restriction sites to the vector ends, but did not have time to try this.
HYPOXIC PROMOTER CHARACTERISATION
While we did not succeed in constructing the hypoxic promoter activity reporter plasmids, we did obtain positive clones for pPEM7-sfGFP and performed a control run to quantify the effect of hypoxia on sfGFP expression to use as a reference for future experiments.
Figure: Difference in sfGFP fluorescence between normoxic and hypoxic conditions
We imaged ~30 individual bacterial cells for each sample and quantified the sfGFP fluorescence intensity within the cell. The variation in intensity between cells in a sample was fairly tight, and the data suggests that sfGFP fluorescence is higher under normoxic conditions than under hypoxic conditions. Statistical analysis using Student’s t-test shows that this difference is significant with a p-value of 0.05, so the promoter-independent variations in sfGFP folding and global protein expression due to hypoxia must still be taken into account while using sfGFP as a reporter for hypoxic assays. Indeed, it has been reported that the sfGFP chromophore formation rate may decrease under hypoxic conditions. The mean decrease in fluorescence observed was 33.93% compared to the normoxic baseline.
