Part:BBa_K731030

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.

Usage and Biology

In cysteine biosynthesis, CysE (a serine acetyltransferase) catalyzes the acetylation of serine to give O-acetylserine, a precursor for the biosynthesis of cysteine. Some O-acetylserine is also converted to N-acetylserine, which in turn triggers the assimilation of sulfate through sulfate assimilation genes.

In Escherichia coli conversion of L-serine to L-cysteine is mediated by the action of two enzymes: serine acetyltransferase [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.
The synthesis of OAS-(thio1)-lyase and of the enzymes involved in sulphate uptake and reduction is regulated by induction as well as by repression [2]. The expression of cysE (the SAT structural gene), on the other hand, is constitutive whereas the catalytic activity of the gene product, SAT, is sensitive to feedback inhibition by L-cysteine [3].

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

The pBAD promoter is active in presence of L-arabinose. L-arabinose binding to the AraC protein inactivates the AraC inhibitory function, permitting RNA polymerase to start transcription of the gene of interest.
AraC is also negatively regulated by cAMP via CRP (formerly known as CAP, catabolite activating protein). In presence of glucose, cAMP levels are low, meaning that AraC can still act as a repressor, not allowing transcription. In the original ara operon, this circuit has the function to limit its transcription when arabinose in not needed, which is when it’s not present and/or when glucose (the primary energy source) is available.

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).

When grown in glucose (MOPS B), cells show an higher O.D. than those grown in glycerol (MOPS A). When induced, cells grow at lower O.D. than the corresponding not induced culture. The pBAD promoter is active in presence of L-arabinose. L-arabinose binding to the AraC protein inactivates the AraC inhibitory function, permitting RNA polymerase to start transcription on cysE, which is controlled by pBAD.
 AraC is also negatively regulated by cAMP via CRP (formerly known as CAP, catabolite activating protein). In presence of glucose, cAMP levels are low, meaning that AraC can still act as a repressor, not allowing transcription. In the original ara operon, this circuit has the function to limit its transcription when no arabinose is present and/or when glucose (the primary energy source) is available. 
When grown in MOPS B cells can use glucose. cysE, which is regulated by araC-pBAD, should not be transcribed, whereas in MOPS A (containing glycerol and no glucose as the carbon source), the protein should be expressed.

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 culture.

Serial dilutions data is confirmative of conclusions coming from growth curves. When grown in MOPS A cultures show a marked decrease in their number. Induction, similarly to what was seen in growth curves, lowers the cell number.

FIGURE 3 Cysteine production upon arabinose induction. Cells were grown as described in Figure 1 and left to grow overnight. A modified version on the assay proposed by Gaitonde et al. was adopted to measure cysteine production in cultures. The 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 Cys, 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 are not expected. In theory, only the culture induced and grow in MOPS A should give an high peak at 560 nm, but in practice it is the opposite. It should be noted that this test didn’t always give the same results during the various attempts performed, probably because of cells growing differently even if the same conditions were maintained. However, even if the absolute intensity of the peaks vary among different experiments, the relative “scale” remains the same. With an high confidence we can affirm that in glucose there is always a good cysteine production, that is often surpassed by the culture grown in glycerol but not induced. A possible explanation of this behavior is that the high expression of the protein when induced and in glycerol (FIg. 21) is, as previously stated, slowing the cells growth, meaning there is not much enzyme in the whole system even if single cells are high producers. On the other hand, the low expression seen in glucose may be counter-balanced or over-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 the two constructs. 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.

WT CysE shows less cysteine production than M256I CysE, as reported in literature [4], in all conditions but when grown in glycerol and induced. WT CysE should not expose the cells to cysteine over-production and over-accumulation as M256I CysE. Hypotetically, WT CysE induction in MOPS A should not have the same imparing consequences as seen with M256I CysE.

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1. EC 2.3.1.30  ↩

2. Jones-Mortimer, 1968; Jones-Mortimer et al., 1968; Kredich, 1971.  ↩

3. Kredich & Tomkins, 1966.  ↩

4. 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