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Sensor: Week 6 June 20-25
From 2011.igem.org
Contents |
Tuesday June 21
Restriction Enzyme Test II
The sensor group digested the K3 vector with the following restriction digests:
| EcoRI | |
| EcoRI | XbaI |
| SpeI | PstI |
| EcoRI | PstI |
| XbaI | SpeI |
These digests were run on an agarose gel. The bands on the gel showed that the restriction enzymes were working.
Wednesday June 22
Radiation Sensor Construct, Day 1
The sensor group continued to assemble their test construct by placing a constitutive promoter in front of two RBS + cI repressor. The sensor group also attempted to place the OR site in front of the test construct pieces (K648000+K648001) and in front of an RBS and reporter. The sensor group tried to clone Cro into K3 and put an RBS in front of the cI repressor.
| Assembly Step | Part Involved with Assembly Step | |
|---|---|---|
| Insert PCR | J23113 B0034+mCherry K648000 | |
| Restriction Digest | Inserts using XbaI and PstI: | B0034+mCherry K648000+K648001 Cro C0051 |
| Insert using EcoRI and SpeI: | J23113 | |
| Vectors using SpeI and PstI: | K3+OR B0032 | |
| Vector using EcoRI and XbaI: | B0031+C0051 | |
| Vector using XbaI and PstI: | K3+RFP | |
| Ligation | OR + B0034+mCherry OR + K648000+K648001 J23113 + B0031+C0051 K3 + Cro B0032 + C0051 J23113 + B0030+C0051 | |
| Transformation/Plating | The above ligations and K3+OR were transformed into Escherichia coli cells. All cells were plated on plates with appropriate resistance. | |
B0034+C0051 and K3 were grown overnight in culture in order to extract more plasmid.
Thursday, June 23
Validate Assemblies from 6/22
The sensor group attempted to validate their assemblies from yesterday through agarose gel electrophoresis.
| Protocol | Part Inolved in Protocol |
|---|---|
| Colony PCR | K3+Cro (Colonies A and B) B0032+C0051 (Colony A) J23113+B0031+C0051 (Colony A) J23113+B0030+C0051 (Colony A) OR+K648000+K648001 (Colony A) OR+B0034+mCherry (Colony A) |
| Agarose Gel Electrophoresis | All colony PCR products above |
| Culture | K3+Cro (colony A) B0032+C0051 (Colony B) J23113+B0031+C0051 (Colony A) J23113+B0030+C0051 (Colony A) OR+K648000+K648001 (colony B) OR+B0034+mCherry (colony A) |
The colonies listed above were cultured because the gel showed that these colonies contained the correct genes. The colonies will be prepared for sequencing through miniprep tomorrow.
Friday, June 24
Sequence Assemblies from 6/22
The plasmids transformed into Escherichia coli cells, which were grown in culture on 6/23, were extracted following the Omega Bio-Tek miniprep protocol. The plasmids were sent to the sequencing center to see if the assemblies worked.
The sensor group also tested their assembly of the OR + K648000 parts through gel electrophoresis. Colony B of this construct was cultured.
Saturday, June 25
The sensor group extracted the OR + K648000 plasmid using the Omega Bio-Tek miniprep protocol. This plasmid will be sent to sequencing on Monday.
