Reporter: Week 4 June 6-11
From 2011.igem.org
Contents |
Monday
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
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
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, GFP, XylE from Registry Parts, Day 1
The reporter group performed PCR to extract tev, 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.
Thursday
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.
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. |