Sensor: Week 8 July 5-8

From 2011.igem.org

Contents

Tuesday, July 5

cI Repressor Test Assembly, Day 27

Sequencing Results: The following constructs were sent to sequencing to verify their DNA sequences:

Construct Sequencing
Result
J23113 + B0034 + C0051 Good
KOR + K648000 Good

Clone OR and CRO

Protocol Part Involved with Protocol
Restriction Digest Inserts using XbaI and PstI: OR
CRO
Vector using XbaI and PstI: K3
Ligation K3 + OR
K3 + CRO
Transformation/Plating The above ligations were transformed into
Escherichia coli cells and plated onto
kanamycin resistant plates.

Wednesday, July 6

cI Repressor Test Assembly, Day 28

The sensor group attempted to place two ribosome binding sites behind the cI repressor.

Protocol Part Involved with Protocol
Insert PCR B0033
B0032
Restriction Digest Inserts using EcoRI and SpeI: B0032
B0033
Vector using 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.

Clone OR and CRO, Take 2

No colonies grew on the kanamycin resistant plates from 7/5, so the cloning was attempted again.

Protocol Part Involved with Protocol
Restriction Digest Inserts using XbaI and PstI: OR
CRO
Vector using XbaI and PstI: K3
Ligation K3 + OR
K3 + CRO
Transformation/Plating The above ligations were transformed into
Escherichia coli cells and plated onto
kanamycin resistant plates.

Thursday, July 7

cI Repressor Test Assembly, Day 29

The sensor group verified their assemblies from 7/6 through agarose gel electrophoresis. The C0051+B0033 looked good, but the C0051+B0032 did not.

Friday, July 8

The C0051+B0033 and K3+CRO constructs were sent to the sequencing center. The C0051+B0033 DNA sequence came back positive, but the K3+CRO did not, so this cloning attempt must be done again.

Since the sequencing results came back positive, freezer stocks were made containing J23113+B0034+C0051 and KOR+K648000.

Colonies of the following constructs were grown up:

KCRO
KOR
B0032+C0051
B0033+C0051

cI Repressor Test Assembly, Day 30

Protocol Part Involved with Protocol
Insert PCR J23113 + B0031 + C0051
J23113 + B0034 + C0051
J2313 + B0030 + C0051
Restriction Digest Inserts using XbaI and PstI: J23113 + B0031 + C0051
J23113 + B0034 + C0051
J2313 + B0030 + C0051
Vector using SpeI and PstI: OR
Ligation OR + K648000 + J23113 + B0031 + C0051
OR + K648000 + J23113 + B0034 + C0051
OR + K648000 + J23113 + B0030 + C0051
Transformation/Plating The above ligations were transformed into Escherichia
coli cells and plated onto kanamycin resistant plates.

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