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Sensor: Week 3 May 30-June 4
From 2011.igem.org
Contents |
Monday
cI Repressor Test Assembly, Day 6
The sensor group ran PCR, transformed, and cultured ribosome binding sites from the registry as well as the cI repressor. The sensor group also prepared for miniprep and sequencing for tomorrow.
| Assembly step | Parts Ivolved with Assembly Step |
|---|---|
| PCR | C0051 from kit |
| Transform/Culture | B0030 B0032 C0051 from kit |
| Colony Growth (for sequencing) | B0031+C0051 B0033+C0051 |
Tuesday
cI Repressor Test Assembly, Day 7
The sensor group performed miniprep from the cultures created 5/30/2011. Two RBS and cI repressor constructs were sent to sequencing while assembly was repeated for the other two RBS and the cI repressor since this assembly from last week failed.
| Assembly Step | Parts Involved with Assembly Step | |
|---|---|---|
| Miniprep | For Sequencing: | B0031+C0051 B0033+C0051 |
| For Assembly: | C0051 B0030 B0032 | |
| Restriction Digest | Insert using XbaI and PstI: | C0051 |
| Vector using SpeI and PstI | B0030 B0032 | |
| Ligation | B0030+C0051 B0032+C0051 | |
| Transformation/Plating | The above ligations were transformed into Escherichia coli cells and plated onto Amp resistant plates. | |
Wednesday
cI Repressor Test Assembly, Day 8
The sensor group placed constitutive promoters in front of the RBS+cI repressor construct and attatched another RBS to the cI repressor.
| Assembly Step | Parts Involved in Assembly Step | |
|---|---|---|
| Restrction Digest | Inserts using XbaI and PstI: | J23113 J23115 C0051 |
| Vectors using SpeI and PstI: | B0033 B0031+C0051 | |
| Ligation | J23113 + B0031+C0051 J23105 + B0031+C0051 B0033 + C0051 | |
| Transformation/Plating | The above ligations were transformed into Escherichia coli cells and plated onto Amp resistant plates. | |
Thursday
cI Repressor Test Assembly, Day 9
The sensor group checked the colonies from the assembly performed on 6/1/11. The sensor group continued their attempt to add two RBS in front of the cI repressor. The sensor group also assembled their parts submitted to the registry (the mCherry reporter construct in front of the cI repressor construct).
| Assembly Step | Parts Involved with Assembly Step | |
|---|---|---|
| Colony PCR | J23113+B0031+C0051 (A) J23105+B0031+C0051 (A) B0033+C0051 (A and B) | |
| Restriction Digest | Insert using EcoRI and SpeI: | K648000 |
| Vectors using EcoRI and XbaI: | K648001 K648002 | |
| Insert using XbaI and PstI: | C0051 | |
| Vectors using SpeI and PstI: | B0030 B0032 | |
| Ligation | K648000 + K648001 K648000 + K648002 B0030 + C0051 B0032 + C0051 | |
| Agarose Gel Electrophoresis | All colony PCR products from above *Results: None of the colonies showed the right number of base pairs. Thus, this part of the assembly must be redone. | |
| Transformation/Plating | The above ligations were transformed into Escherichia coli cells and plated onto Amp resistant plates. | |
Friday
cI Repressor Test Assembly, Day 10
The sensor group checked the colonies from the plates created on 6/2/11 through gel electrophoresis.
| Assembly Step | Part Involved with Assembly Step |
|---|---|
| Colony PCR | K648000+K648001 (A and B) K648000+K648001 (A and B) B0030+C0051 (A and B) B0032+C0051 (A and B) |
| Agarose Gel Electrophoresis | All colony PCR products from above *Results: All colonies showed the correct base pair length so all colonies were cultured for further use. |
Cro and OR Assembly via PCR, Day 1
The sensor group also began assembling the OR and Cro parts of the lambda phage system using the designed primers. The parts were first constructed through PCR, then amplified through a second PCR. The first PCR products were labeled with a 1, and the second PCR products were labelled with a 2.
| Assembly Step | Part Involved with Assembly Step |
|---|---|
| PCR Assembly | Cro OR |
| Agarose Gel Electrophoresis | Cro 1 Cro 2 OR 1 OR 2 *Results: Cro 2 and OR 2 proved to contain the correct base pair length. |
