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Reporter: Week 4 June 6-11
From 2011.igem.org
Contents |
Monday, June 6
Lac Inducible Test Assembly: Take 4, Day 4
The reporter group took the miniprep for the Plac+XylE construct to sequencing. The sequence results showed that the Plac assembled with the first 750 bp of XylE. Thus, the assembly was verified.
Insert Imperial Linker into C3 Vector, Day 2
The reporter group checked to see if the insertion of the Imperial linker into the C3 vector worked.
| Assembly Step | Part Involved with Assembly Step |
|---|---|
| Colony PCR | Imperial linker+C3 |
| Agarose Gel Electrophoresis | The colony PCR product was run on a 1% agarose gel. |
| Culture | 5 ml of SOB media with 6 μl of chloramphenicol was innoculated with the Imperial linker+C3 colony. |
- Results: The gel showed a band less than 100 base pairs which matches the Imperial linker's 85 base pair length.
Extract LacZα from stock part, Day 1
The reporter group ran PCR to knock out the LacZα part from Mike's stock. The agarose gel showed that the PCR product contained the correct number of base pairs.
Tuesday, June 7
Lac Inducible Test Assembly: Take 4, Day 5
The reporter group began making stock of the Plac+XylE.
| Assembly Step | Part Involved with Assembly |
|---|---|
| Transformation | J33204+R0010 was transformed into Escherichia coli cells. |
| Culture | The SOB media and the Amp antibiotic mixture was innoculated with the transformed cells from above. The culture was left shaking overnight. |
Insert Imperial Linker into C3 Vector, Day 3
The Imperial linker+C3 culture was miniprepped and taken to sequencing. The results showed the Imperial linker with a truncated prefix (no XbaI), followed by violacein. Thus, the assembly failed.
Wednesday, June 8
Lac Inducible Test Assembly: Take 4, Day 6
The reporter group prepared freezer stock of the correctly assembled Plac+XylE construct, as well as the following parts from the registry.
| Registry Part | Colloquial Name |
|---|---|
| K327018 | cI+LVA tag |
| K082009 | Lambda repressor |
| K316007 | GFP-XylE Imperial part (for testing) |
| K316017 | Lac controlled tev |
| J33204+R0010 | Plac+XylE with RBS |
Extract tev protease, GFP, XylE from Registry Parts, Day 1
The reporter group performed PCR to extract tev protease, GFP and XylE from their registry parts. The specific parts are listed below:
| Gene of Interest | Registry Part | Length of Gene of Interest (bp) |
|---|---|---|
| K316007: | XylE | 925 |
| K316007: | GFP+flag tag | 800 |
| K316017: | tev protease | 720 |
| Assembly Step | Part Involved with Assembly Step |
|---|---|
| Miniprep | K316007 K316017 |
| PCR | K316007 (twice with different primers, designed May 24) K316017 |
| Agarose Gel Electrophoresis | All above PCR products |
- Results: The gel showed bands all corresponding to 1000 base pairs, which is approximately correct for the genes of interest. Sequencing will further verify our PCR products.
Insert Imperial Linker into K3 Vector, Day 1
Since inserting the Imperial linker into the C3 vector did not work, we tried inserting the Imperial linker into the K3 vector, using the purified PCR product from 6/4/11. We did the insertion through amplified insert assembly.
| Assembly Step | Part Involved with Assembly Step | |
|---|---|---|
| Restriction Digest | Insert using XbaI and PstI: | Imperial Linker |
| Vector using XbaI and PstI: | K3 | |
| Ligation | Imperial linker+K3 | |
| Transformation/Plating | The above ligation was transformed into Escherichia coli cells and plated onto Kanamycin resistant plates to let grow overnight. | |
Mutagenesis of XylE, Take 1 Day 1
The reporter group began the mutagenesis of the XylE gene in order to eliminate restriction sites to allow cloning of the XylE gene into the K3 vector. The reporter group used a slightly modified version of the Knight Lab Multi-site PCR protocol. The extension time used was 2:45.
Thursday, June 9
Insert Imperial Linker into K3 Vector, Day 2
The reporter group checked the 6/8 insertion of the Imperial linker into the K3 vector with a 1% agarose gel.
| Assembly Step | Part Involved with Assembly Step |
|---|---|
| Colony PCR | Two colonies (A&B) of Imperial linker+K3 |
| Agarose Gel Electrophoresis | Above colony PCR product |
| Culture | The Imperial linker+K3 colonies were cultured and allowed to grow overnight. |
- Results: No bands showed up on the agarose gel test. We believe that the DNTPs were labelled incorrectly in the lab because many other PCRs seemed to fail this week. We prepared the colonies for sequencing anyway.
Mutagenesis of XylE, Take 1 Day 2
The reporter group checked to see if the mutagenesis PCR from 6/8 worked. 1 μl of DpnI was added to t hePCR product and incubated at 37° C for one hour. The mixture was then run on a 1% agarose gel. Nothing showed up on the gel, indicating that the mutagenesis process must be repeated.
Insert tev Protease into K3 Vector, Day 1
The reporter group inserted the tev protease into a K3 vector through amplified insert assembly.
| Assembly Step | Part Involved with Assembly Step | |
|---|---|---|
| Restriction Digest | Insert using XbaI and PstI: | tev protease |
| We used the leftover K3 restriction digest from 6/8. | ||
| Ligation | tev protease+K3 | |
| Transformation/Plating | The above ligation was transformed into Escherichia coli cells and plated onto Kanamycin resistant plates. | |
Mutagenesis of XylE, Take 2 Day 1
The reporter group ran the modified Knight lab multi-site PCR protocol, but used two different polymerases: phusion polymerase and phire polymerase. The extension time used was 2:30. The part length was 1845 base pairs and th elength with the plasmid was 3915 base pairs. This information is useful for running the agarose gel electrophoresis. When the group ran the gel, nothing showed in the phusion or the phire lanes. Since the gel electrophoresis proved inconclusive, the reporter group decided to transform their PCR products in order to determine if the mutagenesis worked. After diluting the XylE (1 μl Xyle + 24 μl dH2O), the diluted Xyle, Phusion PCR and Phire PCR were transformed into Escherichia coli cells. These cells were plated onto ampicillin resistant plates.
Friday, June 10
Insert Imperial Linker into K3 Vector, Day 3
The Imperial linker+K3 colonies were miniprepped and sent to sequencing. The sequence results for colony C verified the assembly (It worked!).
Insert tev Protease into K3 Vector, Day 2
The reporter group checked the to see if the insertion of the tev protease into the K3 vector worked.
| Assembly Step | Part Involved with Assembly Step |
|---|---|
| Colony PCR | tev protease+K3 (3:10 extension time) |
| Agarose Gel Electrophoresis | above colony PCR product |
| Culture | The two tev protease+K3 colonies were cultured overnight. |
Results: The gel test showed that the tev protease+K3 construct contained the correct number of base pairs, so the tev protease+K3 construct should be sent to sequencing.
Mutagenesis of XylE, Take 2 Day 2
The following additions were made to the phusion and phire polymerase PCR products:
1 μl DpnI
1 μl AgeI
1 μl NgoMIV
After these additions, the PCR products were left to incubate for four hours at 37<deg>C.
Plating Results: The diluted XylE had colonies that filled the amp resistant plate, but plates of Phusion and Phire polymerase PCR products transformed into Escherichia coli cells had no colonies. Thus, mutagenesis must be performed again.
Mutagenesis of XylE, Take 3 Day 1
The reporter group performed the modified Knight lab multi-site PCR protocol with even more changes:
*Blue font indicates change from modified Knight lab multi-site PCR protocol.
PCR Mixture
- 5 μl 10x Taq ligase buffer
- 5 μl T4 ligase buffer
- 10 μl phusion polyermerase buffer
- 2 μl dNTPs
- 2 μl K316007 miniprep (DNA template)
- 0.5 μl primer 1
- 0.5 μl primer 2
- 0.5 μl primer 3
- 17.5 μl dH2O
- 2 μl reverse primer R
- 1 μl phusion polymerase
- 2 μl T4 PNK
- 2 μl Taq ligase
*These primers were designed Wednesday, May 25.
The PCR products were run on a 1% agarose gel, but no bands showed. Thus, mutagenesis did not work and must be done again. The PCR products were purified, transformed into Escherichia coli cells, and then plated on ampicillin resistant plates. No colonies grew, so mutagenesis needs to be done again.
Saturday, June 11
Insert tev Protease into K3 Vector, Day 3
The culture was miniprepped in order to get sequenced on Monday, 6/13/11.
Mutagenesis of XylE, Take 3 Day 2
DpnI was added to the PCR products from 6/10 and left incubating at 37°C for 5.5 hours, then placed in the 4°C fridge.
