Team:Paris Liliane Bettencourt/Project/Memo-cell/Microcin

From 2010.igem.org

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==What is microcin C?==
==What is microcin C?==
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<br><br>Microcins are a class of small (>10-kDa) antibacterial agents produced by Escherichia
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<br>Microcins are a class of small (>10-kDa) antibacterial agents produced by Escherichia
coli and its close relatives (1, 2, 15). Microcins are produced from ribosomally synthesized
coli and its close relatives (1, 2, 15). Microcins are produced from ribosomally synthesized
peptide precursors. The microcin C (McC) is posttranslationally modified by dedicated
peptide precursors. The microcin C (McC) is posttranslationally modified by dedicated
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<br><br>McC is taken up by E. coli through the action of the Yej- ABEF transporter (11) and is
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<br>McC is taken up by E. coli through the action of the Yej- ABEF transporter (11) and is
processed once it is inside the cell. Processing involves deformylation of the N-terminal
processed once it is inside the cell. Processing involves deformylation of the N-terminal
Met residue by protein deformylase, followed by degradation by any one of the three
Met residue by protein deformylase, followed by degradation by any one of the three
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into sensitive cells, where it is processed with subsequent release of the inhibitory
into sensitive cells, where it is processed with subsequent release of the inhibitory
payload.
payload.
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==Engineering of the microcin C operon.==
==Engineering of the microcin C operon.==
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For our project, we needed to have a inducible death gene, as small as possible (<40bp),
For our project, we needed to have a inducible death gene, as small as possible (<40bp),
so that it could fit within a recombination site but also could be triggered only when
so that it could fit within a recombination site but also could be triggered only when

Latest revision as of 06:32, 27 October 2010


Memo-Cell project: Microcin






What is microcin C?


Microcins are a class of small (>10-kDa) antibacterial agents produced by Escherichia coli and its close relatives (1, 2, 15). Microcins are produced from ribosomally synthesized peptide precursors. The microcin C (McC) is posttranslationally modified by dedicated maturation enzymes encoded by genes in the microcin C operon mccABCDE (15). McC is a heptapeptide with covalently attached C-terminal modified AMP (11). The peptide moiety of McC is encoded by the 21-bp mccA gene, the shortest bacterial gene known (6). McC (Fig. 1) has a molecular mass of 1,178 Da and contains a formylated N-terminal methionine, a C-terminal aspartate instead of the asparagine encoded by the mccA gene, and an AMP residue attached to the carboxamido group of the modified aspartate through an N-acyl phosphoramidate bond. The phosphoramidate group is additionally esterified by a 3-aminopropyl moiety.

How it works?


McC is taken up by E. coli through the action of the Yej- ABEF transporter (11) and is processed once it is inside the cell. Processing involves deformylation of the N-terminal Met residue by protein deformylase, followed by degradation by any one of the three broad-specificity aminopeptidases (peptidases A, B, and N) (9).
Processed McC (Fig. 1) strongly inhibits translation by preventing the synthesis of amino- acylated tRNAAsp by aspartyl-tRNA synthetase (AspRS) (10). The tRNA aminoacylation reaction catalyzed by aminoacyl tRNA synthetases includes two steps. First, the enzyme activates a cognate amino acid by coupling it to ATP and forming aminoacyl-AMP (aminoacyl-adenylate). The aminoacyl moiety is then transferred to tRNA.
Processed McC is structurally similar to aspartyl-AMP but is not hydrolyzable. Thus, the inhibition of AspRS results from tight binding of processed McC in place of aspartyl- adenylate.
Thus, McC is a Trojan horse inhibitor (13): the peptide moiety is required for McC delivery into sensitive cells, where it is processed with subsequent release of the inhibitory payload.

Engineering of the microcin C operon.


For our project, we needed to have a inducible death gene, as small as possible (<40bp), so that it could fit within a recombination site but also could be triggered only when recombination did not happen, that is to say, when the bacteria did not «count» well.
Hence, microcin C seemed to be the only choice as it is only 24bp long.
However, microcin C is naturally produced by one bacteria to survive in a depleted media by killing surrounding bacterias, which is the opposite of what we needed.
We had then to engineer to operon so that once induced, the microcin C would kill the producing bacteria but not the rest of the population.

To do this, we had to tackle three major keypoints:

  • 1. Knock-out the gene coding for the membrane transporter responsible for the uptake of

the microcin.

  • 2. Knock-out genes within the operon which are responsible for self-immunity against the

microcin

  • 3. Mutate the operon so as to delete the four biobrick restriction sites (3 PstI and 1 EcoRI).