Sensor: Week 4 June 6-11

From 2011.igem.org

(Difference between revisions)
(cI Repressor Test Assembly, Day 15)
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===Cro and O<sub>R</sub> Insert into K3 Vector, Day 1===
===Cro and O<sub>R</sub> Insert into K3 Vector, Day 1===
K3 + OR colony A<br />K3 + OR colony B<br />K3 + Cro colony A<br />K3 + Cro colony B
K3 + OR colony A<br />K3 + OR colony B<br />K3 + Cro colony A<br />K3 + Cro colony B
 +
were all cultured in order to have the constructs sequenced.
were all cultured in order to have the constructs sequenced.

Revision as of 16:23, 15 June 2011

Contents

Monday

cI Repressor Test Assembly, Day 12

The minipreps prepared on 6/4 (K648000+K648001, B0030+C0051, B0032+C0051) were sent to sequencing. The sequencing results showed that the K648000+K648001 and B0030+C0051 assemblies worked. However, the B0032+C0051 assembly only produced vector background. Thus, stock should be made of the K648000+K648001 and B0030+C0051 constructs and the B0032+C0051 assembly should be redone.

Cro and OR Insert into C3 Vector, Day 1

The sensor group attempted to insert the Cro and OR parts created through PCR on 6/3 into separate C3 vectors through amplified insert assembly.

Assembly Part Part Involved with Insert Step
Restriction Digest Inserts using XbaI and PstI: OR
Cro
Vector using XbaI and PstI: C3+violacein
Ligation OR+C3
Cro+C3
Transformation/Plating The above ligations were transformed into
Escherichia coli cells and plated onto
Chloramphenicol resistant plates.

Tuesday

cI Repressor Test Assembly, Day 13

The sensor group worked to make stock of their correct assemblies. The following constructs were transformed into Escherichia coli cells and allowed to grow in culture in order to make freezer stock.

K648000+K648001
B0030+C0051
B0034+C0051

Freezer stock was made from the B0031+C0051 culture as well.

Cro and OR Insert into C3 Vector, Day 2

All of the colonies on the plates were purple, meaning that the insert never inserted into the vector. Thus, Cro and OR must be inserted into a different vector.

Wednesday

cI Repressor Test Assembly, Day 14

The B0034+C0051 construct was sent to sequencing. The sequencing results showed that the assembly worked. Freezer stocks were made from the cultures made 6/7 (listed above).

Cro and OR Insert into K3 Vector, Day 1

Since the C3 vector did not accept the Cro and OR inserts, the sensor group attempted to insert Cro and OR into the K3 vector.

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

Thursday

cI Repressor Test Assembly, Day 15

The sensor group attempted to place a constitutive promoter (J23113) in front of the cI repressor and different ribosome binding sites.

Assembly Step Part Involved with Assembly Step
Restriction Digest Insert using EcoRI and SpeI: J23113
B0033
Vector using EcoRI and XbaI: B0030+C0051
B0031+C0051
C0051
Insert using XbaI and PstI: C0051
Vector using SpeI and PstI: B0032
Ligation J23113 + B0030+C0051
J23113 + B0031+C0051
B0033 + C0051
B0032 + C0051
Transformation/Plating The above ligations were transformed into
Escherichia coli cells and plated onto
ampicillin resistant plates.

Cro and OR Insert into K3 Vector, Day 1

K3 + OR colony A
K3 + OR colony B
K3 + Cro colony A
K3 + Cro colony B

were all cultured in order to have the constructs sequenced.

Friday

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