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Sensor: Week 5 June 12-17
From 2011.igem.org
Contents |
Sunday 12 June 2011
cI Repressor Test Assembly, Day 18
The Sensor group ran PCR of the K648000+K648001 using taq polymerase. The PCR products will be inserted into the OR vector.
Monday 13 June 2011
cI Repressor Test Assembly, Day 19
The assemblies that were cultured on 6/10/11 (J23113+B0030+C0051, J23113+B0031+C0051, B0032+C0051 were sent to sequencing. The results showed that only the vector grew, meaning that the assemblies will have to be performed.
The OR site was placed in front of the cI repressor testing construct assembled on Monday, 6/6/11 as well as in front of a terminator and mCherry reporter.
| Assembly Step | Part Involved with Assembly Step | |
|---|---|---|
| Restriction Digest | Insert Using SpeI and PstI: | OR |
| Vectors Using XbaI and PstI: | K648000+K648001 B0034+mCherry | |
| Ligation | OR + K648000+K648001 OR + B0034+mCherry | |
| Transformation/Plating | The above ligations were transformed into Escherichia coli cells and plated onto Kanamycin resistant plates. | |
Tuesday
cI Repressor Test Assembly, Day 20
The sensor group re-assembled two ribosome binding sites + cI repressor with a constitutive promoter as the insert.
| Assembly Step | Part Involved with Assembly Step | |
|---|---|---|
| Restriction Digest | Insert using EcoRI and SpeI: | J23113 |
| Vectors using EcoRI and XbaI: | B0030+C0051 B0031+C0051 | |
| Ligation | J23113 + B0030+C0051 J23113 + B0031+C0051 | |
| Transformation/Plating | The above ligations were transformed into Escherichia coli cells and plated onto Kanamycin resistant plates. | |
Wednesday
cI Repressor Test Assembly, Day 21
| Assembly Step | Part Involved in Assembly Step | |
|---|---|---|
| Restriction Digest | Vector using SpeI and PstI: | K3+OR |
| Inserts using XbaI and PstI: | B0034+mCherry K648000+K648001 | |
| Vector using EcoRI and XbaI: | B0031+C0051 | |
| Insert using EcoRI and SpeI: | J23113 | |
| Ligation | OR + B0034+mCherry OR + K648000+K648001 J23113 + B0031+C0051 | |
| Transformation/Plating | The above ligations were transformed into Escherichia coli cells, then plated onto plates with resistance to the appropriate antibody. | |
The sensor group also ran an agarose gel electrophoresis test on the assembly from 6/14 and the digests from today in order to see the validity of their work.
| Assembly Step | Part Involved with Assembly Step |
|---|---|
| Colony PCR | J23113+B0030+C0051 colonies A and B |
| Agarose Gel Electrophoresis | J23113+B0030+C0051 colony A J23113+B0030+C0051 colony B K648000+K648001 (Digest) B0034+mCherry (Digest) OR (Digest) B0031+C0051 (Digest) J23113 (Digest) |
Gel Results: The agarose gel contained bands corresponding to the correct length of the J23113+B0030+C0051 assembly for colony B. This colony was allowed to grow overnight in culture.
