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From 2011.igem.org

Contents 1 Sunday, June 26 1.1 Insert tev Protease into K3 Vector, Take 7 Day 1 2 Monday, June 27 2.1 Insert tev Protease into K3 Vector, Take 8 Day 1 2.2 Cloning of XylE and GFP, Day 1 2.3 Catechol Assay to Test K316009, Day 1 3 Tuesday, June 28 3.1 Insert tev Protease into K3 Vector, Take 8 Day 2 3.2 Cloning of XylE and GFP, Day 2: Verification 3.3 Catechol Assay to Test K316009, Day 2

Sunday, June 26

Insert tev Protease into K3 Vector, Take 7 Day 1

     The plates containing the assembly from 6/25 grew colonies that only contained RFP, none of which contained the tev protease. Thus, this assembly must be done again.

     The purified tev protease PCR product from 6/4 and the K3 miniprep from 6/25 were digested with XbaI and PstI in buffer 3. The digests were ligated together, then the ligation was transformed into competent Escherichia coli cells and plated onto kanamycin resistant plates.

Monday, June 27

Insert tev Protease into K3 Vector, Take 8 Day 1

     The colonies that grew overnight all contained RFP, meaning that the insertion of tev protease into K3 from 6/26 did not work. As a result, we performed the insertion again. We started with the tev protease (K316017) plasmid from the 6/8 miniprep and the K3 vector miniprep.

Protocol	Part Involved in Protocol
Insert PCR	K316017
Restriction Digest	Insert using XbaI and PstI:	K316017
	Vector using XbaI and PstI:	K3
Ligation	K316017+K3
Transformation/Plating	The ligation above was transformed into Escherichia coli cells then plated on a kanamycin resistant plate.

Cloning of XylE and GFP, Day 1

     The purified PCR products of the mutated XylE and GFP were digested with XbaI and AgeI. The Imperial Linker + K3 served as the vector and was digested with XbaI and AgeI. The two digests were ligated together, transformed into Escherichia coli cells and plated onto kanamycin resistant plates.

Catechol Assay to Test K316009, Day 1

Two 5 ml M9 cultures of K316009 stock were grown up at 37°C with shaking.

Tuesday, June 28

Insert tev Protease into K3 Vector, Take 8 Day 2

     Only one colony was white on the kanamycin resistant plate, meaning that this colony did not contain RFP. This white colony was amplified through colony PCR. The PCR product was run on an agarose gel, which yielded a band less than 500 base pairs long. The tev Protease should be just under 1000 base pairs, so the cloning attempt did not work. One idea was that the problem existed with the insert PCR products (poor purification or loss of product). The PCR product was run on the gel, and the corresponding (bright) band was just under 1000 base pairs, indication that the PCR products are correct and in ample amount, so the problem lies elsewhere.

Cloning of XylE and GFP, Day 2: Verification

     Three colonies of both the XylE + K3 and GFP + K3 and two colonies of RecAI + K3 were amplified through colony PCR. The PCR products were run on an agarose gel, which showed that the constructs contained the correct number of base pairs. The XylE colony B, GFP colony B and RecAI colony A were grown in culture overnight so their plasmids could be extracted for sequencing tomorrow.

Catechol Assay to Test K316009, Day 2

     One of the cultures grown in M9 overnight was induced with 1 μl IPTG (of unknown concentration. Th eother culture was left uninduced. After a one hour incubation at 37°C, 50μl of 10mM catechol was added to each culture. The absorbance (at 380 nm) was recorded over ten minutes. The results were as follows.

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