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Reporter: Week 9 July 10-16
From 2011.igem.org
Contents |
Sunday, July 10
The cleavage sites+GFP colonies were amplified through colony PCR (extension time=1:20). The PCR products were run on an agarose gel, which showed that the tev cleavage site+GFP colony was vector background. The cI cleavage site+GFP contained the correct number of base pairs, so the colony was grown in culture overnight. The sequencing results (which came back on Monday, July 11) showed that the cI cleavage site did not successfully assemble to the GFP vector.
The promoter and RBS construct (J23100+B0034) was placed in front of the GFP+cI cleavage site and in front of the XylE+small linker construct. The same assembly process from Thursday, July 7 was repeated for this assembly.
Monday, July 11
The assembly 25 (openwetware) method for fusion parts was used to place the tev cleavage site in front of GFP. The tev cleavage site insert was cut with EcoRI and AgeI in buffer 1. The GFP vector was cut with EcoRI and NgoMIV in buffer 4. The insert and vector were ligated together, transformed into electrocompetent Escherichia coli cells then plated onto kanamycin resistant plates.
Tuesday, July 12
Only one colony of tev cleavage site+GFP grew overnight. This colony was amplified through colony PCR (extension time=1:25). The PCR products were run on an agarose gel, which yielded a band just above 1000 base pairs. This matched the expected results, so the cells from this colony were grown up in culture overnight. Since only one colony grew, the assembly was repeated as a precaution. This assembly followed the same process as the one performed on Monday, July 11.
The promoter and RBS construct (J23100+B0034) was placed in front of the GFP+tev cleavage site, XylE+small linker and XylE+Imp linker constructs. These assemblies were performed following the same process as the assembly from Thursday, July 7. The transformations had poor time constants (less than 2.0), but the cells were plated on kanamycin resistant plates anyway.
The LacZα and GusA genes were amplified using new primers because the old primers contained stop codons. These genes will be cloned into the K3 vector.
Wednesday, July 13
The amplification of LacZα and GusA were verified along with tev cleavage site+GFP, J23100+B0034+XylE+small linker, J23100+B0034+XylE+Imp linker and the J23100+B0034+GFP+tev cleavage site assemblies using gel electrohporesis. The gel showed that the J23100+B0034+XylE+Imp linker, J23100+B0034+XylE+small linker and tev cleavage site+GFP seemed to work (all contained the correct number of base pairs). The J23100+B0034+GFP+tev cleavage site failed.
The amplification of GusA and LacZα worked, so these two genes were cloned into K3.
| Protocol | Part Involved with Protocol | |
|---|---|---|
| Restriction Digest | Inserts using XbaI and AgeI: | LacZα GusA |
| Vector using XbaI and AgeI: | K3 | |
| Ligation | GusA+K3 LacZα+K3 | |
| Transformation/Plating | The above ligations were transformed into Escherichia coli cells and plated onto kanamycin resistant plates. | |
The transformations had poor time constants (less than 2.0). Something may be wrong with the electrocompetent Escherichia coli cells.
