Team:Heidelberg/Templates/Indigoidine week19 overview

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Revision as of 15:28, 21 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.

@@ Line 1: / Line 1: @@
 ===Domain Shuffling – PPTase Plasmids===
 <p>
-The PPTase plasmids [[Plasmids#pRB15|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.
 </p>
 ===T-Domain Shuffling===
 <p>
-Last week we assembled [[Plasmids#pKH4|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 [[Plasmids#pKH5|pKH5]] in which the T-domain of indC were replaced by ccdB. In parallel and as a backup strategy, we assembled [[Plasmids#pRB19|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/>
-As a second backup strategy, we assembled [[Plasmids#pRB22|pRB22]] and [[Plasmids#pRB23|pRB23]] using [[Methods#CPEC|CPEC]] with single fragments that were amplified from a ''P. luminescens'' cell pellet.
+As a second backup strategy, we assembled pRB22 and pRB23 using CPEC with single fragments that were amplified from a ''P. luminescens'' cell pellet.
 </p>
 <p>
-Both [[Plasmids#pKH5|pKH5]] and [[Plasmids#pRB22|pRB22]] were correct as seen by PCR screenings and sequencing. We used pRB22 to assemble [[Plasmids#pRB23|pRB23]], which is the ccdB-variant of pRB22, enabling to test every combination of T-domain in co-expression with the four PPTases.
+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.
 </p>