Team:Exeter/Results/characterise

Single Gene Plasmids and Enzyme Characterisation - Alex Baldwin

In this mini-project, individual glycosyltransferase genes: WbnJ (BBa_K764000), WbnK (BBa_K764002), WfcA (BBa_K764003), WclY (BBa_K764004) and WbiP (BBa_K764007) have been cloned successfully and sent to the Parts Registry, whereas WbbC (with point mutation) and WbbC were cloned but unable to be sent to the Parts Registry due to time restrictions.

As a continuation of The 3-Gene Inducible Plasmid project, full constructs of: pBAD(large)-RBS-OmpA-SacB-terminator, TetR+RBS-OmpA-SacB-terminator, TetR+RBS-HAS-terminator, pLacI/Ara-1+RBS-WclY-terminator and TetR+RBS-WbnK-terminator were tested for protein expression by adding appropriate inducers (2.5μM L-Arabinose for pBAD(large) promoter, 1μg/mL tetracycline for TetR promoter and 1M IPTG for pLacI/Ara-1 promoter) and identifying novel bands on SDS-PAGE.

No novel bands were detected for HAS, WbnK or WclY on the gel corresponding to the pellet fractions (since all these proteins should stay within the cell) and SacB in the supernatant fractions (since SacB is attached to the signal export sequence, OmpA). However, growth was encountered for E.coli containing the recombinant six constructs, with 5a entering stationary phase and the rest still in exponential phase (see figure 1). Since all six constructs contained the right sequences coding for the correct protein, one possible explanation for these results is that the promoters that were induced were incorrect. This was further proven by the SDS-PAGE gel obtained for samples that came from pelleted fractions, since a whole host of bands were found, except for the novel bands that were expected.

To determine whether the inducible promoters were the issue, another experiment using another SDS-PAGE gel was run using a higher concentration of protein for pBAD(large)-RBS-OmpA-SacB-terminator -inducer/+inducer and TetR+RBS-OmpA-SacB-terminator -inducer/+inducer. These were selected because they both contained OmpA, a new BioBrick which we wanted to submit to the Parts Registry. This would allow other teams to attach this signal peptide to desired proteins so they can be exported out of the cell if needed.

Again, no new bands corresponding to SacB were identified in the supernatant fractions for each full construct. This issue could have resulted from picking penultimate samples which were not induced or grown for as long as the final samples used in the previous experiment that tested protein expression for all six constructs (see figure 1 at 3.0 hours compared to 16.5 hours). Since our results indicated that growth remained high for the experiment involving protein expression of pBAD(large)-RBS-OmpA-SacB-terminator-inducer/+inducer and TetR+RBS-OmpA-SacB-terminator -inducer/+inducer, SacB was clearly not being expressed outside of the cell nor in vivo, as this would have resulted in cell lysis which did not occur.

A related experiment was carried out to determine GFP fluorescence of constructs: pLacI/Ara-1+RBS-WclY-terminator, pLacI/Ara-1+RBS-GFP and pBAD/AraC weak+RBS-GFP. The former construct was used to determine basal levels of GFP fluorescence and each of the three constructs was -induced and +induced. The results of this experiment show that no significant GFP fluorescence was identified (see figure 2).

Taking all these experiments into account, it can be concluded that the promoter(s) are not responding to the appropriate inducer(s). This issue is actually very useful for characterisation of the promoters we have used in our project because we have evidence to suggest that these promoters are not functioning as intended. We will endeavour to update information and any characterisation details on the Parts Registry to help other teams in the future who anticipate to use the same promoters we have used in our project (these being: pBAD/AraC weak, pBAD/AraC(large), pLacI/Ara-1 and TetR).

In this mini-project, it was not possible to conduct SDS-PAGE to determine if the glycosyltransferases would be soluble in the cell and to determine if molecular weights were correct. It was also not possible to undertake glycosyltransferase enzyme assays or mass spectrometry to determine both specificity for each glycosyltransferase for substrate and the overall enzyme kinetics. This is important and necessary for GlycoBase and the modelling aspect of the project and therefore in this mini-project we have planned an enzyme glycosyltransferase assay that will exploit the release of the diphosphonucleotide carrier when the sugar donor bonds with the acceptor sugar.¹

The protocol that would have been used to do the glycosyltransferase enzyme assays would be as follows:

• A 25µL reaction mix containing 10 µL of varying concentrations of donor sugar (0, 100, 200, 300, 400, 500 and 600µM), 10µL 2.5M acceptor sugar and 5 µL 20ng/µL of coupling phosphatase would be added to different wells on a 96-well plate.
• 25µL of 100ng/µL of each individual glycosyltransferase (WbnJ, WbnK, WfcA, WclY, WbbC, WbbC(with point mutation) and WbiP) would then be added to each respective reaction mixes to make a total 50µL volume, and the reactions would then be initiated. A negative control would be used in another well containing 25µL assay buffer instead of the glycosyltransferase solution.
• The 96-well plate would then be sealed and incubated at 30oC for 20 minutes.
• After 20 minutes from initiation of the glycosyltransferase reactions, all reactions would be stopped by adding 30µL of Malachite Green Reagent to each well and mixed gently, followed by adding 100µL of MilliQ H2O to each well and then 30µL of Malachite Green Reagent to each well and mixed gently.
• To produce a distinct colour, all wells would be incubated for 20 minutes at room temperature, followed by reading the colour of each well at 620nm after calibration and subtraction of the negative control reading.
• Measurements of optical density (OD) at 620nm could then be translated to product formation by using a phosphate standard curve since inorganic phosphate release is quantitative to enzyme turnover rate.
• Overall, whilst it was not possible to conduct the enzyme assays or undertake mass spectrometry which were important, this mini-project accomplished the significant cloning work required to undertake such experiments in the future. If time was not an issue, then all glycosyltransferases would be assayed multiple times to determine prinicipally V _max and K_M values to help with the modelling of our system, which in turn would be used to optimise the GlycoBase database. Mass spectrometry would be used to identify both oligosaccharide production and loss of diphosphonucleotide carrier. SDS-PAGE would finally confirm functionality and molecular weight of our glycosyltransferase proteins.

¹R&D systems (2012) Technical Note: A Novel Glycosyltransferase Activity Assay. [Online], Available: here [10^thSeptember 2012].