Reporter: Week 9 July 10-16



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
Vector using XbaI and AgeI: K3
Ligation GusA+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.

Thursday, July 14

     The cI cleavage site+GFP assembly was attempted again, following the process performed on Saturday, July 9. This time, however, the insert restriction digest (EcoRI and AgeI) was reacted in buffer 4 instead of buffer 1.

     The LacZα and GusA cloning into K3 attempt was verified through gel electrophoresis. The gel yielded bands corresponding to the appropriate sizes for LacZα and GusA, but the LacZα was smeared. The smearing was likely a result of running both colony PCRs together, even though GusA (1.8kbp) is much larger than LacZα (300 bp). All of the tested colonies were grown up in culture with the kanamycin antibiotic.

Friday, July 15

Four colonies of the tev mutagenesis attempt were grown up in culture overnight in order to send the plasmids to the sequencing center.

     Somewhere along the line, the cleavage sites were mislabeled. This was noticed when the cleavage sites were used in assemblies, and parts that should have contained cI cleavage site really contained the tev cleavage site, and vice versa. In order to determine the source of the confusion, the original cI and tev cleavage site plasmids, as well as the GFP+cleavage sites plasmids were sent to the sequencing. The sequencing results showed that the cI and tev cleavage site stocks were switched, so these stocks, as well as all tubes containing plasmids that contained the cleavage sites, were relabeled.

Saturday, July 16

The cleavage sites+GFP assemblies were repeated following the same process performed on Thursday, July 14.

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