Difference between revisions of "Part:BBa K731010"

Revision as of 17:31, 24 September 2012

M256I CysE (Serine Acetyltransferase)

CysE is an enzyme involved In cysteine biosynthesis, also known as SAT. It catalyzes the acetylation of serine to give O-acetylserine, the CysE final precursor. Some O-acetylserine is also converted to N-acetylserine, which in turn triggers the assimilation of sulfate through specific genes.

M256I CysE shows enhanced secretion of cysteine and is not inhibited by Cysteine overproduction.

This part has been successfully operated while controlled by araC-pBAD 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 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 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, assimiliting more sulfate to satisfy its needs.

We demonstrated that Part BBa_K731010 works as expected when operated in the Composite Part BBa_K731030, under the control of araC-pBAD (BBa_K731201). NEB10β cells with and without part BBa_K7310130 (i.e. CysE) operated in the low copy vector pSB3C5 were grown in MOPS, supplemented with K2SO4 and glucose as carbon source, and induced with 5 mM Arabinose. After overnight induction a 0.5 mL aliquot was taken from each sample to which it was added 0.5 mL of glacial acetic acid and 0.5 mL of Ninhidrin reagent prepared as described in our protocol page. Panel A: NEB10β cells before induction (left) and after overnight induction (right). Panel B: Cells transformed with BBA_K731030 before induction (left) and after overnight induction (right).

Please head over to K731030 for documentation on characterization of this Part.

Sequence and Features