Sensor: Week 6 June 20-25



Tuesday June 21

Restriction Enzyme Test II

The sensor group digested the K3 vector with the following restriction digests:

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
Restriction Digest Inserts using XbaI and PstI: B0034+mCherry
Insert using EcoRI and SpeI: J23113
Vectors using SpeI and PstI: K3+OR
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
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.

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