Results

benE Transporter

benE transpoters amplified from two different organisms, Rhodococcus sp. and A. baylyi ADP1, were optimised with a His-tag in order to be visually expressed. The expression of the benE protein is shown by a western blot (Figure 1).


Western Blot benE
Figure 1. Lanes 1 & 2 are controls with no induction. Lanes 3 & 5 are benE Rhodococcussp. Lanes 4 & 6 are benE A. baylyi ADP1.


Biosensor

In order to determine that the completed biosensor was functioning as expected a fluorescent assay was performed. The biosensor was transformed into Escherichia coli BW25113 and grown for approximately 20 hours in a CLARIOstar microplate reader and fluorescence data for GFP and mCherry was collected. Data was normalised against uninoculated media controls, meaning fluorescence readings are a result of biosensor activity.


benzoate biosensor
Figure 2. Fluorescent assay of GFP expression. Black data points represent fluorescence in the presence of benzoate while grey represents no benzoate.
Data points are an average of n=3 replicates.



By analysing the data obtained from the fluorescence assay (Figure 2) we were able to determine that the addition of benzoate resulted in an increase in GFP fluorescence. We can therefore determine that the addition of benzoate results in an increase in GFP expression. Comparing the fluorescence of GFP in the presence of benzoate and without allows us to determine that the fluorescence is not just a result of an increasing cell density.


catechol biosensor
Figure 3. Fluorescent assay of mCherry expression. Black data points represent fluorescence in the presence of catechol while grey represents no catechol.
Data points are an average of n=3 replicates.



Similar results were seen when the fluorescence data for mCherry (Figure 3) was analysed. The addition of catechol resulted in an increase in mCherry fluorescence, which we determine to be a result of increased mCherry expression.

Summary

We predict that the increases in fluorescent gene expression seen in this data are a result of upregulation by transcription factors which are responding positively to the presence of the relevant chemical (benzoate or catechol). More testing would be necessary to confirm that this is true.


Cloning the benABCD genes



Initially, we managed to clone the benABCD cluster from A. baylyi and Rhodococcus sp. however, the ribosome binding site was too far from the start codon for us to expect efficient expression. Additionally, the vector used for this cloning was not pTTQRGS6xHisBsaI and thus, we redid the cloning of each cluster and the indivivual genes with new primers and the new vector. Prior to the recloning an expression analysis was performed on BL21 transformed with the cluster from each organism. The total soluble and insoluble protein was extracted from pelleted cells and run on an acrylamide gel which is shown below. There are some differences in the protein bands however, further analysis is required to draw any firm conclusions.

acrylamide gel of total protein


The first step in cloning each of the genes and clusters into the new vector was to amplify the inserts. PCR was used for this with primers designed to introduce the necessary 5' overhang for TEDA cloning. In order to confirm correct amplification the purified PCR products of each of the individual genes was run on a gel as shown below. The PCR was performed in duplicate, with the first set of replicates on the left and the second set on the right. Each of the genes are separated by ladders i.e. benA from A. baylii is followed by benA from Rhodococcus, then a 1kb ladder, then benB from A. baylii and benB from Rhodococcus etc. The bands for each of these genes are all of the expected sizes.

PCR to amplify all genes


PCR was also used to amplify the entire cluster from each organism. The resulting products were also run on a gel to confirm. TEDA cloning was then used to insert the amplified fragments into the pTTQRGS6xHisBsaI vector. The entire solution was used to transform E. coli DH5a. Several colonies were picked for colony PCR in order to confirm the cloning and transformations. The results for A. baylii benA, benB, and benD and also Rhodococcus benA and the benABCD cluster were negative from both colonies picked, so four more colonies were chosen for colony PCR. After both colony PCRs, there was at least one positive colony for each gene and cluster from both organisms. Overnights were made up and the plasmids were miniprepped and digested in order to confirm the fragments had been inserted into the vector. This allowed us to confirm that the cloning had been correct for A. baylii benA, benC, benD and benABCD as well as for Rhodococcus benA, benC and benD. Some of the bands were difficult to make out on the initial gel, and so these were repeated with a lower voltage, longer runtime, and an EtBr stain. The results of both gels are shown below.

Digest of plasmids initial

Digest of plasmids, repeated


The miniprepped plasmids containing these genes were then used to transform colonies of E. coli BL21 for expression experiments.

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