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Sensor: Week 2 May 23-27
From 2011.igem.org
Contents |
Monday
cI Repressor Test Assembly, Day 1
The sensor group began the assembly of a cI repressor testing construct by amplifying the below constitutive promoters via PCR.
| Assembly Step | Parts Involved in Assembly Step |
|---|---|
| PCR | J23113 J23105 J23118 J23100 |
Tuesday
cI Repressor Test Assembly, Day 2
The sensor group continued to build their test construct by inserting the constitutive promoters into the vector containing the cI repressor and ribosome binding site (B0034+C0051). The sensor group also inserted a ribosome binding site and color reporter (B0034+mCherry) in front of a terminator (B0015).
| Assembly Step | Parts Involved in Assembly Step | |
| PCR Purification | J23113 J23105 J23118 J23100 | |
| Miniprep | B0051 | |
| Restriction Digest | Inserts using EcoRI and SpeI: | J23113 J23105 J23118 J23100 B0034+mCherry |
| Vectors using EcoRI and XbaI: | B0034+C0051 B0015 | |
| Ligation | J23113 and B0034+C0051 J23105 and B0034+C0051 J23118 and B0034+C0051 J23100 and B0034+C0051 B0034+mCherry and B0015 | |
| Transformation/Plating | The above ligations were transformed into Escherichia coli cells and plated onto Amp resistant plates. | |
| PCR | B0030 B0031 B0032 B0033 | |
Wednesday
cI Repressor Test Assembly, Day 3
The sensor group inserted various ribosome binding sites (listed below) in front of the cI repressor (C0051). The sensor group also finished the assemblies from yesterday by performing colony PCR on the plated colonies.
| Assembly Step | Parts Involved with Assembly Step | |
|---|---|---|
| Colony PCR | B0034+mCherry+B0015 (Kan resistance) B0034+mCherry+B0015 (Amp resistance) | |
| PCR Purification | B0030 B0031 B0032 B0033 | |
| Restriction Digest | Inserts using EcoRI and SpeI: | B0030 B0031 B0032 B0033 |
| Vector using EcoRI and XbaI: | C0051 | |
| Ligation | B0030+C0051 B0031+C0051 B0032+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 4
The sensor group put the reporting assembly (B0034+mCherry+B0015) in front of two cI repressor construct with different constitutive promoters (J23113+B0034+C0051 and J23105+B0034+C0051).
| Assembly Step | Part Involved with Assembly Step | |
|---|---|---|
| Miniprep for Sequencing | B0034+mCherry+B0015 J23113+B0034+C0051 J23105+B0034+C0051 | |
| PCR | B0034+mCherry+B0015 | |
| Restriction Digest | Insert using EcoRI and SpeI: | B0034+mCherry+B0015 |
| Vectors using EcoRI and XbaI: | J23113+B0034+C0051 J23105+B0034+C0051 | |
| Ligation | B0034+mCherry+B0015 and J23113+B0034+C0051 B0034+mCherry+B0015 and J23105+B0034+C0051 | |
| Transformation/Plating | The above ligations were transformed into Escherichia coli cells and plated onto Amp resistant plates. | |
Note: The minipreps in green font indicate the sequencing proved the assembly worked.
Friday
cI Repressor Test Assembly, Day 5
The week's assemblies have been confirmed and named for the registry.
| Assembly | Registry Name |
|---|---|
| B0034+mCherry+B0015 J23113+B0034+C0051 J23105+B0034+C0051 | K648000 K648001 K648002 |
