Team:ULB-Brussels/Parts

From 2014.igem.org

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$The$ $Mighty$ $Coli$ $Biobrick$
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$The$ $Mighty$ $Coli$ $BioBrick$
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<p> In order to characterize the ccdB biobrick, we sent the biobricks to sequencing and made a <i>screen of activity </i> for the protein ccdB. We did a killing assay, because of the toxic property of ccdB.<br><!--$\small killing$ $\small assay$--><br>
<p> In order to characterize the ccdB biobrick, we sent the biobricks to sequencing and made a <i>screen of activity </i> for the protein ccdB. We did a killing assay, because of the toxic property of ccdB.<br><!--$\small killing$ $\small assay$--><br>
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We constructed 4 different colonies including one with the plasmid pKK-233-ccda, another with pBAD33-ccdB, a third with both, and a control colony. The ccdA gene encoded for a protein wich acts as an anti-toxin of ccdB.<br><!--$\small\hspace{0.08cm} 4$ $\small different$ $\small colonies\hspace{0.06cm}$ --><br>
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We constructed 4 different colonies including one with the plasmid pKK233/ccdA, another with pBAD33/ccdB, a third with both, and a control colony. The ccdA gene encoded for a protein wich acts as an anti-toxin of ccdB.<br><!--$\small\hspace{0.08cm} 4$ $\small different$ $\small colonies\hspace{0.06cm}$ --><br>
On the first media containing <i>IPTG</i> (inducing the pKK233’s expression) <i>and glucose</i> (repressing the pBAD's expression), each colony grew. That allowed us to control the non toxicity of ccdA.
On the first media containing <i>IPTG</i> (inducing the pKK233’s expression) <i>and glucose</i> (repressing the pBAD's expression), each colony grew. That allowed us to control the non toxicity of ccdA.

Revision as of 08:03, 12 October 2014

$~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ \newcommand{\MyColi}{{\small Mighty\hspace{0.12cm}Coli}} \newcommand{\Stabi}{\small Stabi}$ $\newcommand{\EColi}{\small E.coli} \newcommand{\SCere}{\small S.cerevisae}\\[0cm] ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ \newcommand{\PI}{\small PI}$ $\newcommand{\Igo}{\Large\mathcal{I}} \newcommand{\Tgo}{\Large\mathcal{T}} \newcommand{\Ogo}{\Large\mathcal{O}} ~$ Example of a hierarchical menu in CSS

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- Université Libre de Bruxelles -



$The$ $Mighty$ $Coli$ $BioBrick$


Our Part


$\Longrightarrow$ ULB-Brussels part

Properties

Name: BBa_K1318000
Host: E.coli
Source: ccdBA operon
Plasmid: available using LabGenius BioBrick Mapper
Length: 309 bp
Sequence: (begin) atgcagttt ... atataataa (end)
Compatibility: RFC[10], RFC[12], RFC[21], RFC[23], RFC[25].

Characterization

In order to characterize the ccdB biobrick, we sent the biobricks to sequencing and made a screen of activity for the protein ccdB. We did a killing assay, because of the toxic property of ccdB.

We constructed 4 different colonies including one with the plasmid pKK233/ccdA, another with pBAD33/ccdB, a third with both, and a control colony. The ccdA gene encoded for a protein wich acts as an anti-toxin of ccdB.

On the first media containing IPTG (inducing the pKK233’s expression) and glucose (repressing the pBAD's expression), each colony grew. That allowed us to control the non toxicity of ccdA. On the media containing both IPTG and arabinose (inducing the pBAD's expression), the strand with pBAD was killed and the strand with both ccdA $\small\&$ ccdB grew.

We made dilutions to assure that the cell concentration didn’t affect the toxicity or the anti-toxicity.



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