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Reporter: Week 6 June 20-25
From 2011.igem.org
Contents |
Monday
Mutagenesis of XylE, Take 5 Day 5
No growth was seen in any of the three overnight cultures. The most probable reason for the lack of growth in the cultures is the entire colony was used for colony PCR on 6/17, leaving no cells to grow in culture. In order to grow more cells in culture, the three PCR reactions were transformed into competent Escherichia coli cells and plated on chloramphenicol resistant plates.
Insert Small Linker into Imp Linker + K3 Vector, Take 2 Day 1
The small linker and the Imp Linker + K3 construct were digested with XbaI and AgeI in buffer 4. The digests were ligated together, and the ligation was transformed into Escherichia coli cells and plated onto kanamycin resistant plates.
Insert 10 AA Linker into Imp Linker + K3 Vector, Take 3 Day 1
The 10 AA linker and the Imp Linker + K3 construct were digested with XbaI and AgeI in buffer 4. The digests were ligated together, and the ligation was transformed into Escherichia coli cells and plated onto kanamycin resistant plates.
Insert tev Protease into K3 Vector, Take 4 Day 1
No colonies grew from the 6/17 assembly of tev protease and the K3 vector, so the assembly was performed again. The purified tev protease PCR product from 6/4 and the K3 miniprep from 6/13 were digested with XbaI and PstI in buffer 3. The digests were ligated together, and the ligation was transformed into Escherichia coli cells and plated on kanamycin resistant plates.
Tuesday
Mutagenesis of XylE, Take 5 Day 6
All three plates contained ten total colonies:
| PCR Reaction | Number of Colonies | Colonies Used |
|---|---|---|
| 1 | 8 | A, B, C |
| 2 | 0 | N/A |
| 3 | 2 | A, B |
The colonies that did grow were amplified through colony PCR, then tested through agarose gel electrohporesis. This test showed that the PCR reactions were the correct size in base pairs, so the reactions are ready for sequencing. Cultures containing colonies 1A, 1B, 1C, 3A, and 3B were prepared and allowed to grow overnight in the 37°C shaking incubator.
6/20 Assembly Validations
The tev protease + K3 colonies all still contained RFP, which means that the assembly did not work. The small linker + K3 and 10 AA + K3 plates both grew colonies, although not many.
Two colonies from both the small linker + K3 and the 10 AA + K3 plates were amplified through colony PCR. The PCR products were tested through agarose gel elctrophoresis in order to determine their size in base pairs. The gel showed that the two linkers contained the correct number of base pairs, so these constructs should be sent to sequencing.
Wednesday
Mutagenesis of XylE, Take 5 Day 7
The 1A, 1C, 3A and 3B cultures were prepared for sequencing through the Omega Bio-Tek miniprep protocol. The miniprep for culture 1B was contaminated and then abandoned. The other four minipreps were sent to sequencing with the reverse primer. The sequencing results for colony 3A showed all three mutations, meaning that this mutagenesis worked.
6/20 Assembly Sequencing
The 10 AA culture did not grow any cells, most likely because all of the 10 AA colonies were used for colony PCR, leaving nothing to grow in the culture. Thus, the 10 AA linker must be reassembled.
The small linker culture was prepared for sequencing through the Omega Bio-Tek miniprep protocol. The sequencing results showed that the assembly from 6/20 worked. As a result, freezer stock of the small linker was prepared for both the -20°C and -80°C freezers.
