Team:Exeter/Results/characterise

Single Gene Plasmids and Enzyme Characterisation - Alex Baldwin

Currently being edited

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.

Unfortunately, no novel bands were detected for HAS, WbnK, WfcA and WbnJ 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). Another SDS-PAGE gel was run using a higher concentration of protein for pBAD(large)-RBS-OmpA-SacB-terminator and TetR+RBS-OmpA-SacB-terminator and, again, no new bands corresponding to SacB were identified in the supernatant fractions for each full construct. This latter issue could have resulted from picking penultimate samples which were not induced for as long as the final samples used (which were grown overnight) for comparing protein expression of all six full constructs.

However, 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.

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 Vmax and KM 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.