Difference between revisions of "Part:BBa K731030"

Revision as of 22:42, 20 September 2012

Inducible araC-pBAD promoter regulating M256I CysE

The CysE M256I gene (K731010) is here regulated by the araC-pBAD promoter (K731201), which is inducible by arabinose.

This part has been successfully operated both in pSB1C3 (K731030) and the low copy vector pSB3C5, in which it was characterized. A sfGFP tagged fusion of this part has also been deposited as BBa_K731040 and used to test protein expression levels upon arabinose induction.
This part was cloned by the iGEM Trento 2012 team for the creation of an aerobically engineered pathway for the removal of the black crust disfiguring marble stones. Further information about this part and its characterization can be found in the iGEM Trento 2012 wiki.

This Part is also available into the medium copy vector pSB3C5. They are available upon request ( igemtrento [at] gmail [dot] com )

Usage and Biology

CysE is a serine acetyltransferase that mediates the production of cysteine. More specifically, CysE  [1] catalyses the activation of L-serine by acetyl-CoA. Its product, 0-acetyl-L-serine (OAS), is then subsequently converted to L-cysteine by 0-acetyl-L-serine(thio1)lyase [2].
The catalytic activity of CysE is sensitive to feedback inhibition by L-cysteine [3].

Denk and Bock [4], isolated a M256I cysE mutant that had a 10-fold decrease in feedback inhibition by cysteine itself, in the end promoting cysteine secretion into the medium.
This particular mutant, thus, would overproduce cysteine, needing and assimiliting more sulfate to satisfy its needs.

The M256I cysE gene is here regulated by the araCpBAD promoter, which is active in presence of L-arabinose. L-arabinose binds to the AraC protein and inactivates the AraC inhibitory function, permitting to the RNA polymerase to start transcription of the gene of interest (i.e. cysE).
AraC is also negatively regulated by cAMP via CRP (formerly known as CAP, catabolite activating protein). In presence of glucose, cAMP levels are low. This means that AraC can still act as a repressor, not allowing transcription.

FIGURE 1 Effect of M256I CysE in cell growth. Cell density was measured at different time points to determine the effect of M256I CysE expression. Cells were grown at 37°C in LB until it was reached an OD of 0.6. The cells were then spinned down and resuspended in an equal volume of MOPS medium and allowed to grow to an OD of 0.8. Prior the induction cells were splitted into two samples of equal volume. One of the two samples was induced with 5 mM arabinose. Every hour a 0.75mL volume was taken to measure the OD. This assay was performed in two different MOPS media: with 60 mM glycerol (MOPS A) and with 30 mM glucose (MOPS B).

FIGURE 2 Toxicity of M256I CysE in cell growth by serial dilutions. Cultures were grown as described in Figure 1. A sample taken at 4 hours or 8 hours was diluted by a 10², 10³,10⁴, and 10⁵ scale-factor. 150ul of each sample were plated. Colonies were counted the day after to assess the original cell number in each culture.

FIGURE 3 Cysteine production upon arabinose induction. Cells were grown as described in Figure 1 and left to grow overnight. A modified version of the assay proposed by Gaitonde et al. was adopted to measure cysteine production and secreted in the media. The ninhydrin reagent was prepared mixing 250mg of ninhydrin in 10mL of a solution made of glacial acetic acid (60%) and fuming HCl (40%). Solubilization of ninhyrin occured after about 15min of vortexing at max speed. Samples were prepared in glass tubes, mixing 0.5mL of glacial acetic acid, 0.5mL of the culture to be tested and 0.5mL of the reagent previously described. The mixture was left 10min in a 90°C water bath. In presence of Cysteine, ninhydrin makes the solution turn pink/purple in about 3min. Spectra were recorded from 600nm to 400nm, as the characteristic intensity peak for cysteine is at 560nm. Cysteine concentration was calculated referring to a standard curve. From left to right cells expressing K731030 in: MOPSA before induction, MOPS A- arabinose after 16h, MOPS A + arabinose after 16h, MOPS B before induction, MOPS B - arabinose after 16h, MOPS B + arabinose after 16h.

The results from the assay (Fig. 2) were unexpected. In theory, only the culture induced and grown in MOPS supplemented with glycerol should give an high peak at 560 nm, but in practice it is the opposite. Even if the absolute intensity of the peaks varied among different experiments, the relative “scale” remained the same. Our data show that in glucose there was always cysteine production, that is often surpassed by the culture grown in glycerol not induced. A possible explanation of this behavior could be that since the high expression of the protein when induced in glycerol (Fig. 3 in K731040) is affecting cell growth (Fig. 1), the cells don’t have the capabilities to sustain such high cysteine production. On the other hand, the low expression observed in glucose may be counter-balanced by the rapid growth of cells in these conditions. Not inducing the culture while growing in glycerol also seems to be a good balance, in which the leaky expression of the promoter in combination with glucose absence probably leads to high cysteine levels.

FIGURE 4 K731020 vs K731030. Absorbance at 560 nm is compared between wild type and M256I CysE. Samples are, from left to right: MOPS A not induced, MOPS A induced, MOPS B not induced, MOPS B induced. The blue bar is K731020, while the red one is K731030. CysE indicates wt CysE (blue bars), while cysE★ indicates M256I CysE (red bars).

WT CysE (CysE) shows less cysteine production than M256I CysE (cysE★) , as reported in literature [4], in all conditions but when grown in glycerol and induced.

Sequence and Features