Team:Heidelberg/Templates/Indigoidine week17 overview

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<h3>T-Domain Shuffling</h3>
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===T-Domain Shuffling===
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We started inserting new T-Domains into indC to check their functionality. In total we first assembled 19 variants of [[Plasmids#pRB19|pRB19]], which are named pRB19-Txx. The appended three-letter code identifies the respective T-domain or TTE-domain and is listed [[Plasmids#T-TE-domains|here]].  
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We started inserting new T-Domains into indC to check their functionality. In total we first assembled 19 variants of pRB19, which are named pRB19-Txx. The appended three-letter code identifies the respective T-domain or TTE-domain and is listed on our plasmid page in the materials section.  
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Since PCR screenings of [[Plasmids#pRB19|pRB19]] were inconsistent, we started with replacing the T-Domain of [[Plasmids#pRB14|pRB14]], which contains sfp as a PPTase. Screenings and sequencing results showed that assembly and transformation were correct but all colonies except for that on the positive control retained a white phenotype. We tested all the other T-domains on the newly assembled [[Plasmid#pRB19|pRB19]] with every PPTase [[Plasmids#pRB15|pRB15-18]] next week.
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Since PCR screenings of pRB19 were inconsistent, we started with replacing the T-Domain of pRB14, which contains sfp as a PPTase. Screenings and sequencing results showed that assembly and transformation were correct but all colonies except for that on the positive control retained a white phenotype. We tested all the other T-domains on the newly assembled pRB19 with every PPTase pRB15-18 next week.
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===T-Domain Shuffling – PPTase Plasmids===
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<h3>T-Domain Shuffling – PPTase Plasmids</h3>
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As the PPTase plasmids [[Plasmids#pRB15|pRB15-18]] showed red colonies after retransformation of a positively prepped colony, we picked new colonies and prepped them. We will do the co-transformation experiments with the new version of [[Plasmids#pRB15|pRB15]] but with the old ones of [[Plasmids#pRB16|pRB16-18]].
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As the PPTase plasmids pRB15-18 showed red colonies after retransformation of a positively prepped colony, we picked new colonies and prepped them. We will do the co-transformation experiments with the new version of pRB15 but with the old ones of pRB16-18.
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===BioBricks for the Registry===
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<h3>BioBricks for the Registry</h3>
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This week we removed the two internal RFC[10]-cutting sites of indC (EcoRI and SpeI) on [[Plasmids#pRB21|pRB21]] using a [[Methods#CPEC|CPEC]] approach to yield [[Plasmids#pRB22|pRB22]]. The modified indC (''indC*'') was amplified introducing the RFC[10] prefix and suffix ([[Primer#KH21|Primer KH21/22]]). The four PPTases and indC* coding sequences were cloned into pSB1C3 using standard [[Methods#BBaAssembly|BioBrick Assembly]]. <br/>
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This week we removed the two internal RFC[10]-cutting sites of indC (EcoRI and SpeI) on pRB21 using a CPEC approach to yield pRB22]. The modified indC (''indC*'') was amplified introducing the RFC[10] prefix and suffix (Primer KH21/22). The four PPTases and indC* coding sequences were cloned into pSB1C3 using standard BioBrick Assembly. <br/>
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Due to faulty primer design of [[Primer#RB71|RB71]], the resulting plasmid [[Plasmids#pRB22|pRB22]] was incorrect as well. We ordered a corrected version of [[Primer#RB78]] and assembled the plasmids again in the following week.
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Due to faulty primer design of RB71, the resulting plasmid pRB22 was incorrect as well. We ordered a corrected version of RB78 and assembled the plasmids again in the following week.
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Latest revision as of 03:32, 29 October 2013

T-Domain Shuffling

We started inserting new T-Domains into indC to check their functionality. In total we first assembled 19 variants of pRB19, which are named pRB19-Txx. The appended three-letter code identifies the respective T-domain or TTE-domain and is listed on our plasmid page in the materials section.

Since PCR screenings of pRB19 were inconsistent, we started with replacing the T-Domain of pRB14, which contains sfp as a PPTase. Screenings and sequencing results showed that assembly and transformation were correct but all colonies except for that on the positive control retained a white phenotype. We tested all the other T-domains on the newly assembled pRB19 with every PPTase pRB15-18 next week.

T-Domain Shuffling – PPTase Plasmids

As the PPTase plasmids pRB15-18 showed red colonies after retransformation of a positively prepped colony, we picked new colonies and prepped them. We will do the co-transformation experiments with the new version of pRB15 but with the old ones of pRB16-18.

BioBricks for the Registry

This week we removed the two internal RFC[10]-cutting sites of indC (EcoRI and SpeI) on pRB21 using a CPEC approach to yield pRB22]. The modified indC (indC*) was amplified introducing the RFC[10] prefix and suffix (Primer KH21/22). The four PPTases and indC* coding sequences were cloned into pSB1C3 using standard BioBrick Assembly.

Due to faulty primer design of RB71, the resulting plasmid pRB22 was incorrect as well. We ordered a corrected version of RB78 and assembled the plasmids again in the following week.

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