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Team:Heidelberg/Templates/Indigoidine week14 overview
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===T-Domain Shuffling – Assembly of ccdB Plasmids=== | ===T-Domain Shuffling – Assembly of ccdB Plasmids=== | ||
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| - | In the subsequent weeks we wanted to exchange the indC T-domain with several T-domains of other NRPS modules. These are modules derived from the | + | In the subsequent weeks we wanted to exchange the indC T-domain with several T-domains of other NRPS modules. These are modules derived from the Tyrocidine and Delftibactin pathway, from the ''E. coli'' NRPS entF and two NRPS modules of unknown function from ''P. luminescens'' itself. <br/> |
| - | + | pRB11 (derived from pKH1), pRB12 (derived from pKH2) and pRB13 (derived from pRB3) carry the ccdB gene with a RBS instead of their native T-Domain, thus they express an unfunctional indC and the ccdb toxin which kills ''E. coli'' TOP10 cells. We extracted the ccdB gene from pDONR (Invitrogen, [http://www.lifetechnologies.com/order/catalog/product/12536017 pDONR™221]). ''E. coli'' OneShot ccdb survival cells produce an antidote to ccdB and are able to survive upon transformation with the ccdB carrying plasmid. After exchange of the ccdB gene with a novel T-domain only positive ''E. coli'' TOP10 transformants survive and the background colonies will be diminished. | |
</p> | </p> | ||
<p> | <p> | ||
Assembly and/or transformations were inefficient, as blue colonies on each plate suggest that an excess of template was contained in the transformation mix. <br/> | Assembly and/or transformations were inefficient, as blue colonies on each plate suggest that an excess of template was contained in the transformation mix. <br/> | ||
| - | A ccdB PCR screening of | + | A ccdB PCR screening of pRB12 was positive but transformation in DH5alpha, TOP10 and OneShot ccdb survival cells showed that the ccdB gene is unfunctional. In the following, all the insert from pDONR was used including a promoter and the ccdA gene. <br/> |
Also we used a two plasmid strategy to test every possible combination of engineered indigoidine synthetases and the respective PPTases. We worked with indC on pSB1C3 and the PPTases on separate pSK3K3 derived plasmids. | Also we used a two plasmid strategy to test every possible combination of engineered indigoidine synthetases and the respective PPTases. We worked with indC on pSB1C3 and the PPTases on separate pSK3K3 derived plasmids. | ||
</p> | </p> | ||
Latest revision as of 15:20, 21 October 2013
T-Domain Shuffling – Assembly of ccdB Plasmids
In the subsequent weeks we wanted to exchange the indC T-domain with several T-domains of other NRPS modules. These are modules derived from the Tyrocidine and Delftibactin pathway, from the E. coli NRPS entF and two NRPS modules of unknown function from P. luminescens itself.
pRB11 (derived from pKH1), pRB12 (derived from pKH2) and pRB13 (derived from pRB3) carry the ccdB gene with a RBS instead of their native T-Domain, thus they express an unfunctional indC and the ccdb toxin which kills E. coli TOP10 cells. We extracted the ccdB gene from pDONR (Invitrogen, pDONR™221). E. coli OneShot ccdb survival cells produce an antidote to ccdB and are able to survive upon transformation with the ccdB carrying plasmid. After exchange of the ccdB gene with a novel T-domain only positive E. coli TOP10 transformants survive and the background colonies will be diminished.
Assembly and/or transformations were inefficient, as blue colonies on each plate suggest that an excess of template was contained in the transformation mix.
A ccdB PCR screening of pRB12 was positive but transformation in DH5alpha, TOP10 and OneShot ccdb survival cells showed that the ccdB gene is unfunctional. In the following, all the insert from pDONR was used including a promoter and the ccdA gene.
Also we used a two plasmid strategy to test every possible combination of engineered indigoidine synthetases and the respective PPTases. We worked with indC on pSB1C3 and the PPTases on separate pSK3K3 derived plasmids.
