Difference between revisions of "Part:BBa K731030"

Revision as of 20:44, 26 October 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 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 [1].
The catalytic activity of CysE is sensitive to feedback inhibition by L-cysteine [2].

Denk and Bock [3], 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.

Protein expression levels have been monitored with the sfGFP tagged composite part BBa_K731040.

Effect of CysE on cell growth

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 aliquot 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. 150 ul of each sample were plated. Colonies were counted the day after to assess the original cell number in each culture. This assay was performed in two different MOPS media: with 60 mM glycerol (MOPS A) and with 30 mM glucose (MOPS B).

Enzymatic activity of CysE

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 250 mg of ninhydrin in 10 mL 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 0.5 mL of glacial acetic acid, 0.5 mL of the culture to be tested and 0.5 mL of the reagent previously described. The mixture was left 10 min in a 90 °C water bath. In presence of Cysteine, ninhydrin makes the solution turn pink/purple in about 3 min. Spectra were recorded from 600 nm to 400 nm, 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: MOPS A before induction, MOPS A - arabinose after 16 h, MOPS A + arabinose after 16 h, MOPS B before induction, MOPS B - arabinose after 16 h, MOPS B + arabinose after 16 h. MOPS A: medium supplemented with 60 mM glycerol; MOPS B: medium supplemented with 30 mM glucose.

The results from the assay described in Fig. 3 were unexpected. The levels of cysteine produced are in contrast with the expression levels that were observed using part BBa_K731040, a fluorescently tagged CysE. We demonstrated infact that in the presence of glucose the expression of CysE is significantly decreased, due to glucose inhibitory effect of araCpBAD (for these data see K731040). However, our data show that in glucose there was always cysteine production, that is often surpassed by the culture grown in glycerol but not induced. A possible explanation of this behavior could be the limiting number of cells survived after the high expression, that was found in cultures grown in MOPS medium supplemented with glycerol (see Fig. 2). To test the expression of CysE K731040 was used.

WT CysE vs M256I CysE

FIGURE 4 Comparison between cysteine levels produced by WT CysE and M256I CysE (K731020, K731030). Absorbance at 560 nm is compared between WT and M256I CysE induced overnight cell cultures by ninhydrin assay as described in Fig. 3. 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. MOPS A: medium supplemented with 60 mM glycerol; MOPS B: medium supplemented with 30 mM glucose.

Assembling of an aerobic sulfate reduction pathway

This Part, M256I CysE (BBa_K731030), was also operated together with CysDes (K731400) to engineer bacteria to aerobically reduce sulfate and remove the sulfate component of the black crust from marbles, proving the ability of CysE to reduce sulfate.

FIGURE 5. Cysteine degradation by CysDes (K731400). Cells cotransformed with CysE and CysDes were grown as described in Fig. 1 and induced with 5 mM arabinose and 0.1 mM IPTG as annotated in the legend. Cysteine levels were measured with a ninhydrin assay as described in Fig. 3.

Removal of the black crust

FIGURE 6. Removal of the black crust from a synthetically generated gypsum layer: Scanning Electron Microscopy analysis of the black crust before and after bacterial treatment. Untreated synthetic crust (A), SEM analysis of material scraped from untreated synthetic crust at 500x (B) and 1000x magnification (C). Synthetic crust treated with NEB10β cells expressing CysE and CysDes (Parts BBa_K731030 and BBa_K731400) (D); SEM analysis of material scraped from treated synthetic crust at 500x (E) and 1000x magnification (F).

More info on the removal of the black crust by Parts BBa_K731030 and BBa_K731400 can be found on iGEM UNITN-Trento Project Page.

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1. Jones-Mortimer, M.C.(1968b). The control of sulphate reduction in Escherichia coli by O-acetyl-l-serine. Biochem. J. 106, 33P..  ↩

2. Kredich & Tomkins, 1966. The enzymic synthesis of L-cysteine in Escherichia coli and Salmonella typhimurium. Biochem. J. 241, 4955-4965P.  ↩

3. Denk, D., and A. Bock. 1987. L-cysteine biosynthesis in Escherichia coli: nucleotide sequence and expression of the serine acetyltransferase (cysE) gene from the wild-type and a cysteine-excreting mutant. J. Gen. Microbiol. 133:515–525.  ↩

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BamHI site found at 1144
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal AgeI site found at 979
  Illegal AgeI site found at 1909
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal SapI site found at 961