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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. Since the sequencing results showed that the PCR reaction 3 yielded the correct mutagenesis, freezer stock from culture 3A was prepared for both the -20°C and the -80°C freezers.
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.
Insert 10 AA Linker into Imp Linker + K3 Vector, Take 4 Day 1
The 10 AA linker PCR product from 6/4 and the Imp Linker + K3 miniprep from 6/21 were digested with XbaI and AgeI restriction enzymes in buffer 4. The two digests were ligated together, and the ligation was transformed into competent Escherichia coli cells and plated onto kanamycin resistant plates.
Insert tev Protease into K3 Vector, Take 5 Day 1
The purified tev protease PCR product from 6/4 and the K3 miniprep prepared today were digested with XbaI and PstI restriction enzymes in buffer 3. The digests were ligated together, and the ligation was transformed into competent Escherichia coli cells and plated onto kanamycin resistant plates.
Thursday
6/22 Assembly Validation
The plates containing the tev protease + K3 and the 10 AA linker + K3 assemblies both contained colonies. Two colonies for each construct(A and B for 10 AA linker and C and D for tev protease) were amplified through colony PCR. The PCR products were tested using an agarose gel electrophoresis test to see their respective sizes in base pairs. Nothing showed up on the gel because the forward primer was never added to the PCR reaction mixtures. Each colony was cultured anyway to grow cells that can be sequenced tomorrow.
Catechol Assay, Day 1
A 5 ml culture of M9 medium + chloramphenicol was innoculated with K316009 stock on 6/21 and incubated for about 24 hours. The culture was left on the bench at room temperature for another 24 hours. The culture was induced with 1 μl of IPTG (unknown concentration) and incubated for about 2 hours at 37°C with shaking.
After induction and incubation, 100 μl of 1 mM catechol solution was added to the entire culture.
Results: Almost immediately, the culture began turning from a white color to a yellow color. After vortexing and a few minutes at room temperature, the entire culture was a bright yellow color(reminiscent of lemon-lime Gatorade). Now that we know that this works, we need to run more standardized tests with controls to measure pigment production quantitatively using a spectrophotometer.
Friday
6/22 Assembly Sequencing
Both the 10 AA linker +K3 and the tev protease + K3 cultures were prepared for sequencing using the Omega Bio-Tek miniprep protocol then sent to sequencing.
Results:The 10 AA sequencing results showed that the assembly worked, but the tev protease assembly did not, according to the sequencing results.
