2.6 KiB
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I. Coding sequence of ccdB.
ccdB encodes toxic protein (CcdB), which acts as a DNA gyrase toxicant, locking the DNA gyrase together with broken double-stranded DNA, ultimately promoting bacterial suicide. In our initial design, this gene has been optimized and adapted to our chassis strain E. coli MG1655 at the time of synthesis.
II. Coding sequence of btuB.
btuB is an outer membrane porin that mediates high affinity binding and TonB- dependent active transport of vitamin B12 (cyanocobalamin) across the outer membrane. This gene has been optimized and adapted to our chassis strain E. coli MG1655 at the time of synthesis.
III. Coding sequence of araBAD promoter.
araBAD promoter is an E. coli promoter that is tightly controlled by inducer L-arabinose and repressor Arac.
IV. Coding sequence of Fusion araBAD promoter with btuB and rrnB T2 terminator.
ParaBAD system for induction of btuB expression by the arabinose operon 1-6 bp: Xba I site; 7-291 bp: araBAD promoter 315-2159: coding sequence of btuB 2160-2187 bp: rrnB T2 terminator 2188-2193 bp: Xho I site
V. Coding sequence of Expression of AraC.
Expression of repressor AraC. The dual function of the AraC protein as a positive and negative regulator of PBAD activity is achieved through two isoforms of the protein. 'Pr' is a repressor form that can bind to a now unidentified manipulation-like site, and 'Pi' is an inductive form that regulates by binding to the promoter PBAD. In the absence of arabinose, the 'Pr form predominates; Once arabinose is present, it is able to bind to the AraC protein, moving the equilibrium toward the 'Pi' form so that the induction of arabinose can be interpreted as arabinose binding to 'Pr', moving 'Pr' out of its binding site, and then, producing a large amount of 'Pi', which binds to the promoter.
1-157 bp: araC promoter 158-169 bp: RBS 170-1048: coding sequence of araC 1049-1135 bp: rrnB T1 terminator