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Sensor: Week 7 June 28-July 1
From 2011.igem.org
Contents |
Tuesday, June 28
cI Repressor Test Assembly, Day 23
The sensor group continued their assembly of their testing construct. They placed an RBS in the K3 vector with Cro, the OR in front of the mCherry reporter, terminator, cI repressor and J23113 promoter as well as in front of the 34mCherry. Finally, the sensor group attempted (again) to put the B0032 RBS in front of the cI repressor.
| Protocol | Part Involved with Protocol | |
|---|---|---|
| Restriction Digest | Inserts using XbaI and PstI: | 34mCherry K648000+K648001 |
| Inserts using EcoRI and SpeI: | B0032 B0034 | |
| Vectors using EcoRI and XbaI: | C0051 K3+Cro | |
| Vector using SpeI and PstI: | K3+OR | |
| Ligation | B0034 + K3 + Cro KOR + K648000 + K648001 KOR + 34mcherry B0032 + C0051 | |
| Transformation/Plating | The above ligations were transformed into Escherichia coli cells and plated on kanamycin and ampicillin resistant plates. | |
The sensor group also transformed K3+Cro and J23113+B0031+C0051 in order to make stocks. The B0034+C0051 was grown up from stock.
Wednesday, June 29
cI Repressor Test Assembly, Day 24
The sensor group verified their assemblies from 6/28. Colonies containing KOR + 34mcherry, B0034 + K3 + Cro, KOr + K648000+K648001 were amplified through colony PCR. Agarose gel electrophoresis showed that these assemblies seemed to work. The colonies were grown up in culture so the plasmids can be extracted and sent to the sequencing center tomorrow morning.
The B0034 and B0034+C0051 plasmids were extracted using the Omega-Biotek miniprep protocol. These plasmids will be used at a later date.
Thursday, June 30
cI Repressor Test Assembly, Day 25
The following constructs made on 6/28 were sent to sequencing.
KOr + 34mcherry
B0034 + K3 + Cro
KOr + K648000 + K648001
The results showed that these colonies were just vector background. As a result, these assemblies must be done again.
The sensor group also assembled a constitutive promoter to two RBS+cI repressor part. Also, the KOR part was placed in front of the mCherry+terminator part.
| Protocol | Part Involved with Protocol | |
|---|---|---|
| Restriction Digest | Inserts using EcoRI and SpeI: | J23113 B0032 B0033 |
| Insert using XbaI and PstI: | K648000 | |
| Vectors using EcoRI and XbaI: | B0034 + C0051 C0051 | |
| Vector using SpeI and PstI: | KOR | |
| Ligation | J23113 + B0032 + C0051 J23113 + B0033 + C0051 KOr + K648000 | |
| Transformation/Plating | The above ligations were transformed into Escherichia coli cells and plated on kanamycin and ampicillin resistant plates. | |
The cI repressor (C0051) plasmid was transformed into Escherichia coli cells from the registry kit plate.
The following constructs were sent to sequencing:
| Construct | Sequencing Result |
|---|---|
| J23113 + B0032 + C0051 | good |
| J23113 + B0033 + C0051 | bad |
| KOr + K648000 | good |
Friday, July 1
cI Repressor Test Assembly, Day 26
The sensor group tried attaching the cI repressor to two different RBS.
| Protocol | Part Involved with Protocol | |
|---|---|---|
| Restriction Digest | Inserts with EcoRI and SpeI: | B0032 B0033 |
| Vector with EcoRI and XbaI: | C0051 | |
| Ligation | C0051 + B0032 C0051 + B0033 | |
| Transformation/Plating | The above ligations were transformed into Escherichia coli cells and plated onto ampicillin resistant plates. | |
