"

From 2011.igem.org

Contents 1 Sunday 1.1 Mutagenesis of Xyle, Take 3 Day 4 2 Monday 2.1 Insert tev Protease into K3 Vector, Day 4 2.2 Insert Imperial Linker into K3 Vector, Day 4 2.3 Mutagenesis of Xyle, Take 4 Day 1 2.4 Insert cI and tev Cleavage Sites into K3 Vector, Day 1 3 Tuesday 3.1 Mutagenesis of XylE, Take 4 Day 2 3.2 Insert cI and tev Cleavage Sites into K3 Vector, Day 2 3.3 Mutagenesis of XylE, Take 5 Day 1 3.4 Insert Small and 10 AA Linkers into Imp Linker + K3 Vector, Day 1

Sunday

Mutagenesis of Xyle, Take 3 Day 4

     The PCR product of the mutated XylE gene from the 4°C fridge was purifed, transformed into Escherichia coli cells and plated onto ampicillin resistant plates.

Monday

Insert tev Protease into K3 Vector, Day 4

     Sequencing Results: The tev protease sequence came back 99% correct. The assembly features a single point mutation at base pair 515: TCT (serine) -> CCT (proline).

Insert Imperial Linker into K3 Vector, Day 4

     Since we failed to make freezer stock of this correctly assembled part, we transformed the Imp linker + K3 miniprep made for sequencing on Friday, 6/10 into Escherichia coli cells and plated onto kanamycin resistant plates.

Mutagenesis of Xyle, Take 4 Day 1

     The modified Knight Lab Multi site PCR protocol outlined on Friday, 6/10 was followed for the mutagenesis of the XylE gene. No product was seen on the gel, meaning that the mutagenesis did not work. An alternate PCR for mutagenesis protocol, which contained the following changes from the protocol outlined on Friday, 6/10, was performed.

Material Added	Listed Volume Added on 6/10(μl)	Specifically Modified Volume Added (μl)
primer 486	0.3	0.5
primer 837	0.3	0.5
dH2O	17.5	18

NOTE: This PCR reaction only includes two primers, listed above, because these primers have similar annealing temperatures (79.54°C and 79.38°C) while the third primer has a higher annealing temperature (84.32°C).

Insert cI and tev Cleavage Sites into K3 Vector, Day 1

     Since the Imp linker part in the K3 vector contains all of the restriction sites (EcoRI, XbaI, PstI, SpeI, AgeI) the cI and tev cleavage sites were inserted into the K3 vector using the Imp linker + K3 part. The Imp linker + K3 miniprep created Friday, 6/10 and the purified cI and tev cleavage site PCR products from 6/3 were digested with the XbaI and AgeI restriction enzymes in buffer 4. The cI cleavage site and the tev cleavage site were ligated with the Imp linker + K3 construct. These ligations were transformed into Escherichia coli cells and plated onto kanamycin resistant plates.

Tuesday

Mutagenesis of XylE, Take 4 Day 2

     The reaction performed on 6/13 showed no product on the agarose gel The miniprep of K316007 was run on the gel as well in order to see if the problem with the mutagenesis was the miniprep. The miniprep contained the correct number of base pairs, so the miniprep is not the problem.

Insert cI and tev Cleavage Sites into K3 Vector, Day 2

     Although few colonies grew on the plates overnight, we decided to run colony PCR on the colonies that did grow in order to determine how many base pairs the constructs contained. A gel electrophoresis test showed that both of the assemblies contained the correct number of base pairs (88 base pairs each). Thus, the colonies were allowed to grow overnight in culture for sequencing tomorrow.

Mutagenesis of XylE, Take 5 Day 1

     Three different PCR reactions were performed, all based on the modified Knight Lab protocol.

Reaction 1:

In this reaction, 16 μl of dH₂O was used as well as 1.5 μl of DMSO in the reaction mixture. All other additions to the mixture were the same as outlined on Friday, 6/10.

Reaction 2:

In this reaction, 17 μl of dH₂O was used as well as 0.5 μl of MgSO₄ in the reaction mixture. All other additions to the mixture were the same as outlined onFriday, 6/10

Reaction 3: In this reaction, 15.5 μl of dH₂O and 0.5 μl of MgSO₄ were added to the reaction mixture. All other additions were the same as outlined on Friday, 6/10.

One final reaction was performed that featured 0.5μl of primer 315 (primer 1) and 18.5μl of dH₂O. The other two primers were not added to this reaction mixture.

A gel electrophoresis test showed bands for the reaction 1 listed above, and very faint bands for the other reactions. Since bands finally showed on the gel, all of the PCR reaction products were purified and digested with 1 μl of AgeI, NgoMIV and DpNI. After the addition of restriction enzymes, the digests were left to incubate at 37&degC for 6 hours.

Insert Small and 10 AA Linkers into Imp Linker + K3 Vector, Day 1

     The Imp linker vector and the two inserts (small and 10AA linkers) were both digested with XbaI and AgeI in buffer 4. The vector was ligated with each of the two inserts. These ligations were transformed into Escherichia coli cells and plated onto kanamycin resistant plates.

Back to Notebook