Difference between revisions of "Team:Sydney Australia/Sense"

The first ethylene-oxidising bacterium, Mycobacterium sp. strain E20 , was isolated in 1978 (de Bont and Harder, 1978). Subsequently, the characterisation of the enzymes involved in aerobic ethylene metabolism was aimed at utilising such strains for microbial bioremediation of chlorinated ethenes, such as vinyl chloride, as many of these are environmental pollutants (Mattes et al., 2010). However, a separate stream of research has identified another potential use for ethylene-oxidising bacteria – in regulating and monitoring fruit ripening.

As the key hormone responsible for the ripening of most fruits, ethylene levels need to be monitored at every step of the fruit packaging process, and there is a promising market for a cheap, portable and convenient ethylene sensor. Hence, we turned to Mycobacterium NBB4 , the best characterised ethylene-oxidising bacterium, with the aim of designing a cell-based biosensor for ethylene.

In Mycobacterium NBB4 , the enzymes required for ethylene metabolism are contained within an operon organised in two bidirectional gene clusters. One gene cluster encodes the catabolic enzymes required to convert ethylene into acetyl-CoA, the final product of the degradation pathway which is subsequently directed to the tricarboxylic acid cycle. The other gene cluster encodes the enzymes responsible for synthesising coenzyme M, a cofactor required for the function of the second enzyme in the metabolic pathway.

We had a twofold curiosity to understand and characterise the EtnR1/2 system better, and also utilise it as ethylene-responsive circuitry. In order to do this, we needed to:

• Test recombinant expression of EtnR1 and EtnR2 in E. coli.
• Characterise the interactions between ethylene, EtnR1, EtnR2 and the putative promoter

We would first determine whether the BL21 DE3 strain of E. coli. could support the expression of our two mycobacterial genes. After confirming expression of EtnR1/2 and affinity purification, we can decipher the precise protein-DNA, protein-protein and protein-substrate interactions that make up our system.

This would serve as a proof of concept for a novel regulatory mechanism of ethylene degradation in Mycobacterium NBB4 and other mycobacterial strains.

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).

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.

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 (Figure 8) as part of our wet lab collaboration with 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.

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.