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<div class="boxRight"> <div class = "placeholderbox" style=" background-color: transparent; background-color: transparent; background-repeat: no-repeat; background-image: url(https://static.igem.org/mediawiki/2016/b/b0/T--Sydney_Australia--GelShiftFigure.png); height: 250px;" </div> </div> <br> <p> <b> Figure 9. </b> Electrophoretic mobility shift assay for EtnP DNA. The 250 bp region of EtnP was PCR amplified and 100 ng of DNA incubated with 6 mg of protein. The protein-DNA mixtures were run on a 3% agarose gel at 150 V for 100 minutes and stained with Gel Green overnight for visualisation. Lane 1: NEB 100 bp ladder. Lane 2: EtnP DNA only. Lane 3: EtnR1 protein only. Lane 4: EtnR1 protein and EtnP DNA. Lane 5: BSA protein and EtnP DNA. </p></div> | <div class="boxRight"> <div class = "placeholderbox" style=" background-color: transparent; background-color: transparent; background-repeat: no-repeat; background-image: url(https://static.igem.org/mediawiki/2016/b/b0/T--Sydney_Australia--GelShiftFigure.png); height: 250px;" </div> </div> <br> <p> <b> Figure 9. </b> Electrophoretic mobility shift assay for EtnP DNA. The 250 bp region of EtnP was PCR amplified and 100 ng of DNA incubated with 6 mg of protein. The protein-DNA mixtures were run on a 3% agarose gel at 150 V for 100 minutes and stained with Gel Green overnight for visualisation. Lane 1: NEB 100 bp ladder. Lane 2: EtnP DNA only. Lane 3: EtnR1 protein only. Lane 4: EtnR1 protein and EtnP DNA. Lane 5: BSA protein and EtnP DNA. </p></div> | ||
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+ | <div class="boxAll"> <h2> Next Steps </h2><br> | ||
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+ | Given more time, we would like to fully investigate the rest of the proposed regulatory system - specifically the interaction between EtnR2 and ethylene, and the interaction between EtnR1 and EtnR2. We would plan to use a phosphorylation assay and a pulldown assay to test these respectively, as outlined in Figure 5. <br> <br> Leading on from our characterisation of EtnR1, we would aim to confirm that it acts as a transcription factor, and investigate whether it acts as a positive or negative regulator. Lastly, we would like to examine the strengths of putative promoters from other ethylene-metabolising strains of Mycobacterium, and determine whether the NBB4 system is homologous in other strains. | ||
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Revision as of 00:44, 20 October 2016
Progress and Results
Cloning
We generated coding sequences of EtnR1 and EtnR2 either native to Mycobacterium NBB4 or codon optimised for E. coli. K12 which were then cloned into the pET28c plasmid to be under the control of the T7 promoter . All expression assembly was successful and verified by colony PCR, restriction digestion and Sanger sequencing. We also cloned EtnR1, EtnR2 and the putative promoter from NBB4 into the assembly standard BioBrick pSB1C3 for parts submission verified using similar processes.
Expression assay in BL21 (DE3) E. coli.
To test expression of EtnR1 and EtnR2, we transformed the pET constructs into BL21 (DE3) E. coli. containing a pGro7 helper plasmid expressing the GroEL and GroES chaperone proteins for aiding protein folding. We selected for Km and Cm resistant colonies, induced protein expression upon addition of IPTG in liquid culture and ran the cellular lysates on SDS-PAGE.
Figure 6. SDS-PAGE sizing of EtnR1 and EtnR2 expressed following 6 hour IPTG induction. Cellular lysates were isolated from BL21 (DE3) E. coli. with pGro7 chaperone plasmid expressing pET-EtnR1 or pET-EtnR2, and separated electrophoretically on 12% SDS-PAGE. The bands indicated in red were trypsin-digested and analysed using mass spectrometry. (a) Isolated EtnR1 lysates. (b) Isolated EtnR2 lysates.
EtnR1 was recovered from the soluble lysate fraction at a size of roughly 66 kDa, whereas EtnR2 expression was only detected upon solubilisation with SDS at a size of roughly 25 kDa. This indicates that EtnR2 may be membrane associated or expressed in inclusion bodies. Expression of the two genes was found to be stronger using codon optimised sequences, and so were used for all subsequent applications including protein characterisation and biosensor construction.
Mass spectrometry analysis was performed to confirm the suspected SDS-PAGE bands were our proteins of interest (Figure 1). The amino acid sequences obtained indicated we had successfully expressed EtnR1 and EtnR2 in E. coli. from the native Mycobacterium host (Figure 7).
EtnR1
EtnR2
Finally, EtnR1 and EtnR2 expression with an N-terminal His6-Tag was confirmed via Western blotting using an anti-His antibody performed by the UNSW iGEM team as part of our wet lab collaboration thorough bands observed at the expected sizes for each protein.
RESULT: The Mycobacterium NBB4 genes EtnR1 and EtnR2 were successfully expressed in E. coli. verified through SDS-PAGE, mass spectrometry and Western blot.
Figure 8. Western blot with anti-His antibody. Cellular lysates were isolated from BL21 (DE3) E. coli. with pGro7 chaperone plasmid following IPTG-induced protein expression, and separated using SDS-PAGE. The proteins were transferred to a nitrocellulose blot and probed with anti-His antibody. Lane 1, SeeBlue2 Protein Standard; Lane 6, SDS-solubilised EtnR2 lysate; Lane 7, soluble EtnR2 lysate; Lane 8; SDS-solubilised EtnR1 lysate; Lane 9, soluble EtnR1 lysate; Lane 10, positive control.
Characterisation
In order to isolate purified EtnR1 and EtnR2, we used affinity chromatography based off the attraction between pyridine moieties in histidine residues, and divalent metal cations such as nickel(II) and cobalt(II). Following multiple attempts using Ni2+-loaded columns, we successfully obtained EtnR1 and EtnR2 in purer fractions using Co2+-coated beads, again confirmed via mass spectrometry.
Subsequently, an electrophoretic mobility shift assay (EMSA) was performed to determine the interaction between EtnR1 and EtnP, with band retardation observed when the EtnR1-EtnP complex was run on an agarose gel. This confirms that EtnR1 acts as a DNA-binding protein for EtnP, and provides evidence of its role in transcriptional activation of ethylene metabolism in Mycobacterium NBB4 and other ethylene-oxidising bacteria.
RESULT: EtnR1 was characterised as binding to EtnP, and so functions as a likely transcription factor for regulating ethylene metabolism.
Figure 9. Electrophoretic mobility shift assay for EtnP DNA. The 250 bp region of EtnP was PCR amplified and 100 ng of DNA incubated with 6 mg of protein. The protein-DNA mixtures were run on a 3% agarose gel at 150 V for 100 minutes and stained with Gel Green overnight for visualisation. Lane 1: NEB 100 bp ladder. Lane 2: EtnP DNA only. Lane 3: EtnR1 protein only. Lane 4: EtnR1 protein and EtnP DNA. Lane 5: BSA protein and EtnP DNA.
Next Steps
Given more time, we would like to fully investigate the rest of the proposed regulatory system - specifically the interaction between EtnR2 and ethylene, and the interaction between EtnR1 and EtnR2. We would plan to use a phosphorylation assay and a pulldown assay to test these respectively, as outlined in Figure 5.
Leading on from our characterisation of EtnR1, we would aim to confirm that it acts as a transcription factor, and investigate whether it acts as a positive or negative regulator. Lastly, we would like to examine the strengths of putative promoters from other ethylene-metabolising strains of Mycobacterium, and determine whether the NBB4 system is homologous in other strains.
References
1. de Bont, J. A. M. and Harder, W. (1978). Metabolism of ethylene by Mycobacterium E20. FEMS Microbiology Letters, 3, pp.89-93.
2. Mattes, T. E., Alexander, A. K. and Coleman, N. V. (2010). Aerobic biodegradation of the chloroethenes: pathways, enzymes, ecology, and evolution. FEMS Microbiology Reviews, 34, pp.445-475.
3. Coleman, N. V., Yau, S., Wilson, N. L., Nolan, L. M., Migocki, M. D., Ly, M., Crossett, B. and Holmes, A. J. (2011). Untangling the multiple monooxygenases of Mycobacterium chubuense strain NBB4, a versatile hydrocarbon degrader. Environmental Microbiology Reports, 3(3), pp.297-307.
4. Li, C. (2006) Soluble diiron monooxygenases: the linkage between genotype and phenotype in Mycobacterium NBB4. (Honours thesis) University of Sydney, Sydney, Australia.
5. Stock, A. M., Robinson, V. L. and Goudreau, P. N. (2000). Two-component signal transduction. Annual Review of Biochemistry, 69, pp.183-215.