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Team:Heidelberg/Templates/Indigoidine week19 overview
From 2013.igem.org
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| - | + | <h3>Domain Shuffling – PPTase Plasmids</h3> | |
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The PPTase plasmids pRB15-18 have been reassembled with pSB3K3 from a different plate of the partsdistribution, using a CPEC approach. This week we validated those plasmids. Additionally, we assembled them by restriction cloning. | The PPTase plasmids pRB15-18 have been reassembled with pSB3K3 from a different plate of the partsdistribution, using a CPEC approach. This week we validated those plasmids. Additionally, we assembled them by restriction cloning. | ||
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| - | + | <h3>T-Domain Shuffling</h3> | |
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Last week we assembled pKH4, which is a pSB1C3 derived plasmid coding for indC but lacking a PPTase. We validated the plasmid and used it as the template to assemble pKH5 in which the T-domain of indC were replaced by ccdB. In parallel and as a backup strategy, we assembled pRB19 using restriction cloning. <br/> | Last week we assembled pKH4, which is a pSB1C3 derived plasmid coding for indC but lacking a PPTase. We validated the plasmid and used it as the template to assemble pKH5 in which the T-domain of indC were replaced by ccdB. In parallel and as a backup strategy, we assembled pRB19 using restriction cloning. <br/> | ||
Latest revision as of 03:33, 29 October 2013
Domain Shuffling – PPTase Plasmids
The PPTase plasmids pRB15-18 have been reassembled with pSB3K3 from a different plate of the partsdistribution, using a CPEC approach. This week we validated those plasmids. Additionally, we assembled them by restriction cloning.
T-Domain Shuffling
Last week we assembled pKH4, which is a pSB1C3 derived plasmid coding for indC but lacking a PPTase. We validated the plasmid and used it as the template to assemble pKH5 in which the T-domain of indC were replaced by ccdB. In parallel and as a backup strategy, we assembled pRB19 using restriction cloning.
As a second backup strategy, we assembled pRB22 and pRB23 using CPEC with single fragments that were amplified from a P. luminescens cell pellet.
Both pKH5 and pRB22 were correct as seen by PCR screenings and sequencing. We used pRB22 to assemble pRB23, which is the ccdB-variant of pRB22, enabling to test every combination of T-domain in co-expression with the four PPTases.
