Part:BBa_K2695012
T7 pcrA from A.suillum
Usage and Biology
Perchlorate reductase is an enzyme used mainly by perchlorate reducing bacteria to break down perchlorate into chlorite - a reaction which takes place in the periplasm of the bacterium.
PcrB forms a periplasmic heterodimer with PcrA and comprises the active complex of perchlorate reductase. PcrC is a C-type cytochrome that connects the AB complex to the periplasmic membrane, allowing for electron shuffling. PcrD is predicted to be a molybdenum chaperone protein specific to assembling the pcrABC system. (Youngblut et. al.,2016)
This part was taken from the Dissimilatory Perchlorate Reducing Bacterium (DPRB) Azospira suillum. Formerly known as Dechlorosoma suillum (Coates, Achenbach, 2004), A. suillum has been described as a model organism for studying perchlorate reduction as its metabolism is well characterised and its genome is readily available. Moreover, the genus Azospira is thought to be one of the most ubiquitous of the perchlorate reducing bacteria - samples have been found as far as Antarctica (Coates, Achenbach, 2004)! Perchlorate reduction in A. suillum, including the activity of perchlorate reductase, is only active under these conditions (Chaudhuri, O’Connor. et al. 2002):
- Oxygen concentrations less than 2 mg/l (preferably anaerobic conditions)
- Absence of nitrate
- Presence of perchlorate
Exeter iGEM 2018 chose to use the coding sequence for perchlorate reductase from A. suillum because its extensieve characterisation, including the kinetic parameters of the enzyme. The DNA sequence for chlorite dismutase was also taken from this organism.
Sequence and Features
- 10INCOMPATIBLE WITH RFC[10]Illegal XbaI site found at 65
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BamHI site found at 2864
- 23INCOMPATIBLE WITH RFC[23]Illegal XbaI site found at 65
- 25INCOMPATIBLE WITH RFC[25]Illegal XbaI site found at 65
Illegal AgeI site found at 338
Illegal AgeI site found at 1415 - 1000COMPATIBLE WITH RFC[1000]
Cloning
The T7 promoter from pET21(Novagen) was inserted upstream of the pcrA coding sequence flanked by the biobrick prefix and suffix. This was synthesized by IDT. The biobrick assembly method was used to build the entire pcrABCD operon from BBa K2695012(PcrA) and BBa K2695011 (PcrBCD).
This image shows successful digests of the individual parts and the pSB1C3 backbone. Bands were excised from the gel and using a Promega Wizard gel extraction kit, the DNA was purified. The three parts were combined in a ligation reaction with T4 DNA ligase (ThermoFisher). Transformation into DH5α, growth of overnight cultures followed by DNA miniprep extraction yielded DNA that was sent for Sanger sequencing. This confirmed, to our great surprise and joy, that we had successfully managed to build the complete operon.
Western Blot
To demonstrate enzyme expression a Western Blot was performed. The Blot membrane was probed with an anti-His-tag primary antibody(PcrA) raised in mouse, and an anti-mouse secondary antibody, raised in goat, conjugated to alkaline phosphatase.
Figure 5: Western blot for Pcr constructs
Expected molecular weights (kDa): PcrA = 106.21 | PcrC = 26.81 | PcrD = 26.67
Unfortunately, this image does not show any band corresponding to the molecular weight of PcrA. We know that pcrA must be transcribed (due to the presence of PcrC and PcrD), therefore either PcrA is not translated, is mis-folded and bound up in the cell debris (after BugBusting protocol), or is rapidly degraded.
References
Kostan, J. Sjöblom, B. (2010) Structural and functional characterisation of the chlorite dismutase from the nitrite-oxidizing bacterium “Candidatus Nitrospira defluvii”: Identification of a catalytically important amino acid residue. Journal of Structural Biology, Vol 172 (issue 3), pp 331 - 342 [online]
Coates, J D. Achenbach, L A. (2004) Microbial perchlorate reduction: rocket-fuelled metabolism. Nature Reviews Microbiology, Vol no. 2 pp 569–580. [online]
Chaudhuri, S K. O’Connor, S M. Gustavson, R L. Achenbach, L A. Coates, J D. (2002) Environmental Factors That Control Microbial Perchlorate Reduction. Applied and Environmental Microbiology. Vol no. 68 (9) pp 4425–4430. [online]
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