Reporter: Week 5 June 12-17

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Latest revision as of 17:26, 13 July 2011

Sunday, June 12

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, June 13

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.

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, June 14

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

     Four 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.

Reaction 4:

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.

Testing Imperial Construct, Day 1

A stock of M9 medium was made up by adding 50 ml 5X M9 salt solution to 200 ml of water and autoclaving on liquid for 20 minutes. The following additions were made after the autoclave finished:

• 11.25 ml dH₂O
• 1 gram glucose
• 0.5 Casamino Acid
• 1.25 mg MgSO₄ 7-hydrate
• 75 mg Thiamine HCl
• 1.25 ml CaCl₂ solution

Wednesday, June 15

Mutagenesis of XylE, Take 5 Day 2

     Each of the PCR reaction products from 6/14 were amplified following the standard insert PCR protocol. The reactions were then run on another 1% agarose gel. The gel contained single bands around 3000 base pairs for PCR reactions 1, 2 and 3, but not for reaction 4. The PCR products from reaction 1, 2 and 3 were all transformed into Escherichia coli cells and plated onto ampicillin resistant plates.

Insert cI and tev Cleavage Sites into K3 Vector, Day 3

     The cI and tev cleavage sites + K3 constructs were sent to sequencing. The results showed that these assemblies worked, and freezer stock were prepared of these parts.

Insert Small and 10 AA Linkers into Imp Linker + K3 Vector, Day 2

     Nothing grew on the 10 AA linker plate, and only one viable colony grew on the small linker plate. The small linker was amplified through colony PCR with a 45 second extension time. The PCR product was run on a 1% agarose gel, which showed that the small linker contained the correct number of basepairs. The small linker colony was put into culture to grow overnight.

Insert tev Protease into K3 Vector, Take 2 Day 1

The purified tev protease (K316017) PCR product created 6/4 was digested with XbaI and PstI in buffer 3. The K3 restriction digest created 6/8 was used for ligation with the tev protease insert. The ligation was transformed into Escherichia coli cells and plated onto kanamycin resistant plates. However, no colonies grew, so the assembly would have to be done again.

Thursday, June 16

Mutagenesis of XylE, Take 5 Day 3

The XylE mutants were replated on chloramphenicol resistant plates. All previous plating had been on ampicillin resistant plates, but should have been plated on the chloramphenicol resistant plates. This is why this part of the project recieved so much trouble growing colonies.

Insert cI and tev Cleavage Sites into K3 Vector, Day 4

Sequencing Results: The sequencing results showed that the two cleavage site assemblies worked. Thus, freezer stock was made for these parts.

Insert Small and 10 AA Linkers into Imp Linker + K3 Vector, Day 2

     The small linker was prepared for sequencing through miniprep, then was sent to sequencing. The results showed that the assembly did not work, and must be performed again. The small linker sequencing results were not available until late in the day, so the next try at assembling the small part and the K3 vector will be done tomorrow.

Insert 10 AA Linker into Imp Linker + K3 Vector, Take 2 Day 1

     The Imp linker + K3 vector and the 10 AA linker were digested with XbaI and AgeI in buffer 4. The digests were ligated together, and the ligation was transformed into Escherichia coli cells, which were plated onto kanamycin resistant plates.

Prepare 100 mM Catechol Solution

2.202 g of catechol was placed into 200 ml of dH₂O and stored with a tin foil lid to protect from light in the 4°C.

Friday, June 17

Mutagenesis of XylE, Take 5 Day 4

     A few colonies grew on each of the plates from the 3 PCR reactions. One colony from reaction one, two colonies from reaction two and three colonies from reaction three were run on a 1% agarose gel. The gel showed that all the colonies contained the correct number of base pairs. The colonies were placed into SOB media and allowed to grow overnight in culture.

Insert tev Protease into K3 Vector, Take 3 Day 1

     The tev protease (K316017) insert and the K3 vector were digested with XbaI and PstI in buffer 3. The two digests were ligated together. The ligation was transformed into Escherichia coli cells, which were plated onto kanamycin plates and allowed to grow over night.

Insert 10 AA Linker into Imp Linker + K3 Vector, Take 2, Day 2

No colonies grew on the plates, so this assembly needs to be performed again.

Testing Imperial Construct, Day 2

The culture grown in M9 on 6/15 was incubated at 37°C for about 20 hours while shaking, then left on the bench at room temperature for one day. The OD₆₀₀ was measured at 0.661. A stock of K316007 was made and stored in both the -20°C and the -80°C freezers.

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