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Reporter: Week 10 July 17-23
From 2011.igem.org
Sunday, July 17
No colonies grew for tev cleavage site+GFP, and only one colony grew for cI cleavage site+GFP. This colony was amplified through colony PCR (extension time=1:20). The PCR products were run on an agarose gel, which yielded a band that corresponded to around 1000 bp, which was expected. The colony was grown up in culture overnight.
Monday, July 18
The tev mutagenesis and cI cleavage site+GFP plasmids were sent to the sequencing center. The sequencing results showed that the cI cleavage site+GFP worked, but the tev mutagenesis did not.
Since we have had trouble inserting the cleavage sites in front of the GFP gene, the GFP was inserted behind the cleavage sites.
| Protocol | Part Involved with Protocol | |
|---|---|---|
| Insert PCR (extension time=1:15) | GFP | |
| Restriction Digest | Insert using NgoMIV and PstI (buffer 1) | GFP |
| Vector using AgeI and PstI (buffer 1) | ||
| Ligation | tev cleavage site+GFP | |
| Transformation/Plating | The above ligation was transformed into Escherichia coli cells and plated on kanamycin resistant plates. | |
The promoter and RBS construct (J23100+B0034) was placed in front of GFP+tev cleavage site, XylE+small linker and XylE+Imp linker following the procedure performed on Thursday, July 7.
The GusA and LacZα genes were cloned into K3 using the 10AA linker+K3 as the vector.
Tuesday, July 19
The promoter and RBS colonies that started growing on Monday, July 18 were amplified through colony PCR (extension time=1:15). The PCR products were run on an agarose gel, which yielded bands that corresponded to the correct number of base pairs. These colonies were grown up in culture. The J23100+B0024+XylE+10AA linker and J23100+B0024+GFP+cI cleavage site cultures were frozen as stocks.
The GusA and LacZα genes were cloned into K3 following the same procedure performed on Monday, July 18.
The linkers were placed in front of XylE.
| Protocol | Part Involved with Protocol | |
|---|---|---|
| Restriction Digest | Insert using NgoMIV and PstI (buffer 1): | XylE |
| Vector using AgeI and PstI (buffer 1): | 10 AA linker Imp linker small linker | |
| Ligation | 10AA linker+XylE Imp linker+XylE small linker+XylE | |
| Transformation/Plating | The above ligations were transformed into Escherichia coli cells and plated on kanamycin resistant plates. | |
Results: The 10 AA liker+XylE worked, but the Imp linker+XylE and small liker+XylE must be repeated.
