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

From 2010.igem.org

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==What is microcin C?==
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<br><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
 +
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.
 +
<br><br></html>
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==How it works?==
 +
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 +
<br><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
 +
Met residue by protein deformylase, followed by degradation by any one of the three
 +
broad-specificity aminopeptidases (peptidases A, B, and N) (9).
 +
<br>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.
 +
<br>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.
 +
<br>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.
 +
<br></html>
 +
==Engineering of the microcin C operon.==
 +
<html><br><br>
 +
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.
 +
<br>Hence, microcin C seemed to be the only choice as it is only 24bp long.
 +
<br>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.
 +
<br>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.
 +
 +
<br><br>To do this, we had to tackle three major keypoints:
 +
</html>
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*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).

Revision as of 06:30, 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).