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From 2011.igem.org

Contents 1 Sunday, July 17 2 Monday, July 18 3 Tuesday, July 19 4 Thursday, July 21 5 Friday, July 22

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):	tev cleavage site
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.

Thursday, July 21

The promoter and RBS assembly from Wednesday, July 20, was verified. Four colonies were amplified through colony PCR. The PCR products were run on an agarose gel, which showed bands that corresponded to the expected sizes. The colonies were grown up in culture.

The sequencing results for the GusA and LacZα cloning attempts from Monday, July 18 and Tuesday, July 19 showed that GusA was missing the NgoMIV and AgeI sites. Also, both LacZα and GusA were cut in the wrong place, resulting in the appearance of only part of the correct sequence. Nexte time, separate insert PCRs will be run with higher annealing temperatures.

Friday, July 22

The cleavage sites+GFP constructs were placed behind the J23100+B0034+XylE+linker constructs.

Protocol	Part Involved with Protocol
Insert PCR (extension time=1:20)	tev cleavage site+GFP cI cleavage site+GFP
Restriction Digest	Inserts using NgoMIV and PstI (buffer 1):	tev cleavage site+GFP cI cleavage site+GFP
	Vectors using AgeI and PstI (buffer 1):	J23100+B0034+XylE+small linker J23100+B0034+XylE+Imp linker J23100+B0034+XylE+10AA linker
Ligation	J23100+B0034+XylE+small linker+tev cleavage site+GFP J23100+B0034+XylE+small linker+cI cleavage site+GFP J23100+B0034+XylE+Imp linker+tev cleavage site+GFP J23100+B0034+XylE+Imp linker+cI cleavage site+GFP J23100+B0034+Xyle+10AA linker+tev cleavage site+GFP J23100+B0034+XylE+10AA linker+cI cleavage site+GFP
Transformation/Plating	The above ligations were transformed into Escherichia coli cells and plated onto kanamycin resistant plates.

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