Part:BBa_K2626002
RBS + todE
Translational unit consisting of a strong ribosome binding site (RBS) and the enzyme todE. TodE belongs to a group of enzymes called catechol-2,3-dioxygenases (C23Os). TodE specifically encodes for the enzyme 3-methylcatechol 2,3-dioxygenase. This enzyme is part of a series of enzymes responsible for the degradation of aromatic compounds such as toluene, xylene and benzene and their incorporation into the cell metabolism as a carbon source. It is naturally most effective and responsible for the initial cleavage of the 3-methylcatechol intermediate to produce the meta fission product (MFP) 2-hydroxy6-oxo-methylhexa-2,4-dienoate (6-methyl HODA).
Figure 1: The important residues in the TodE enzyme identified included F187, H241 and Y250. These are the ones that seem to have the most interaction with 3-methylcatechol.
However, because the pathway isn't highly specific for toluene, similar aromatic metabolites such as styrene (the monomer of the plastic polystyrene) can be recognised by the toluene operon. TodE has been seen to take in the intermediate of styrene breakdown 3-vinylcatechol, however at high concentrations of 3-vinylcatechol todE is inactivated due to accidental oxidation of active-site iron Fe(II)to Fe(III) (George et al., 2010). During our project, due to the unavailabilty of 3-vinylcatechol, 3-methylcatechol was used to characterise the function of todE, whilst overexpression was attempted to overcome inactivation by the presence of more enzyme.
To characterise todE, an adapted version of the asay used by George et al., 2010 was used. A control curve was created of 3-methylcatechol in lysis buffer due to its tendancy to degrade in room temperature and gain a yellow colour over time. This standard curve was deducted from the assay results that were obtained at the same time. This standard curve allowed us to visualise the assay results more reliably and only see the increase of absorbance at 360 nm only due to the activity of todE.
Figure 2: 3-methylcatechol control curve obtained from parallel reading with assay samples.
In twice-repeated samples, 2mM of 3-methylcatechol was added toBL21 E. coli carrying todE as a control and a RBS + todE transfected BL21 E. coli 24h lysate samples. These samples were read every 30 minutes and the following graph was obtained. The results were normalised against the 3-methylcatechol control curve.
Figure 3: Graph showing the increase of absorbance values of todE control and RBS+todE wells over 30 minute intervals after the addition of 3-methylcatechol.
By analysing the results, we can conclude that after deducting the 3-methylcatechol control, the RBS+todE well drastically increased in absorbance. Also, the todE containing wells also show a steady increase of absorbance at 360 nm. This would confirm that there is a reaction occuring in the wells that we believe is to be the breakdown of 3-methylcatechol to make the MFP 6-methyl HODA, that in fact is the reason the wells turn slightly yellow over time. To support this, an SDS-PAGE gel was run with lysed control, todE and RBS+todE samples from times 0, 6 and 24 after protein induction with IPTG.
Figure 4: SDS PAGE gel with control, todE and RBS+todE translational unit lysis samples.
By studying figure 3, a band can be seen to appear at todE and RBS+todE samples between 25 and 32 kDa indicated by red arrows. We can assume that due to the absence of these bands in any of the control samples as well as referring to George et al., 2010 and the SDS-PAGE results they have obtained that also identify the todE band, we have effectively expressed the todE enzyme. Also, upon further inspection, the suspected RBS+todE bands are much darker and larger, with the t=6 sample being more visible than the band seen on todE sample t=24.
This would further support the assumption that todE is being overexpressed due to the presence of the RBS upstream of the plasmid, supported by the darker bands seen in the SDS-PAGE gel, as well as the assay results showing that the absorbance reading of RBS+todE is 3 and a half times larger than todE at time interval of 210 minutes.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 365
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal AgeI site found at 415
- 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI.rc site found at 440
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