Sensor: Week 5 June 12-17

From 2011.igem.org

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 +
==Tuesday==
 +
===cI Repressor Test Assembly, Day 20===
 +
     The sensor group re-assembled two ribosome binding sites + cI repressor with a constitutive promoter as the insert.
 +
{|border="1"
 +
!Assembly Step
 +
|colspan="2" align="center"|'''Part Involved with Assembly Step'''
 +
|-
 +
|rowspan="2"|Restriction Digest
 +
|Insert using EcoRI and SpeI:
 +
|J23113
 +
|-
 +
|Vectors using EcoRI and XbaI:
 +
|B0030+C0051<br />B0031+C0051
 +
|-
 +
|Ligation
 +
|colspan="2"|J23113 + B0030+C0051<br />J23113 + B0031+C0051
 +
|-
 +
|Transformation/Plating
 +
|colspan="2"|The above ligations were transformed into Escherichia<br />coli cells and plated onto Kanamycin resistant plates.
 +
|}
 +
==Wednesday==
 +
===cI Repressor Test Assembly, Day 21===
 +
{|border="1"
 +
!Assembly Step
 +
|colspan="2" align="center" |'''Part Involved in Assembly Step'''
 +
|-
 +
|rowspan="4"|Restriction Digest
 +
|Vector using SpeI and PstI:
 +
|K3+O<sub>R</sub>
 +
|-
 +
|Inserts using XbaI and PstI:
 +
|B0034+mCherry<br />K648000+K648001
 +
|-
 +
|Vector using EcoRI and XbaI:
 +
|B0031+C0051
 +
|-
 +
|Insert using EcoRI and SpeI:
 +
|J23113
 +
|-
 +
|Ligation
 +
|colspan="2"|OR + B0034+mCherry<br />OR + K648000+K648001<br />J23113 + B0031+C0051
 +
|-
 +
|Transformation/Plating
 +
|colspan="2"|The above ligations were transformed into<br />Escherichia coli cells, then plated onto plates<br />with resistance to the appropriate antibody.
 +
|}
 +
The sensor group also ran an agarose gel electrophoresis test on the assembly from 6/14 and the digests from today in order to see the validity of their work.
 +
{|border="1"
 +
!Assembly Step
 +
!Part Involved with Assembly Step
 +
|-
 +
|Colony PCR
 +
|J23113+B0030+C0051 colonies A and B
 +
|-
 +
|Agarose Gel<br />Electrophoresis
 +
|align="center"|J23113+B0030+C0051 colony A<br />J23113+B0030+C0051 colony B<br />K648000+K648001 (Digest)<br />B0034+mCherry (Digest)<br />OR (Digest)<br />B0031+C0051 (Digest)<br />J23113 (Digest)
 +
|}
 +
''Gel Results:'' The agarose gel contained bands corresponding to the correct length of the J23113+B0030+C0051 assembly for colony B. This colony was allowed to grow overnight in culture.
 +
==Thursday==
 +
===cI Repressor Test Assembly, Day 22===
 +
The J23113+B0030+C0051 colony B culture was prepared for sequencing through the Omega Bio-Tek miniprep protocol.
 +
 +
Because many of the assemblies throughout the lab have failed to yield any colonies on the plates, the restriction enzymes were tested. The B0034+C0051 construct was digested with:
 +
{|
 +
|EcoRI+SpeI
 +
|-
 +
|EcoRI+XbaI
 +
|-
 +
|XbaI+PstI
 +
|-
 +
|SpeI+PstI
 +
|-
 +
|XbaI+SpeI
 +
|-
 +
|EcoRI+PstI
 +
|}
 +
These digests were tested using the agarose gel electrophoresis test, but the results were inconclusive.
[[Team:Penn_State/Notebook| Back to Notebook]]
[[Team:Penn_State/Notebook| Back to Notebook]]

Latest revision as of 16:45, 28 June 2011

Contents

Sunday 12 June 2011

cI Repressor Test Assembly, Day 18

The Sensor group ran PCR of the K648000+K648001 using taq polymerase. The PCR products will be inserted into the OR vector.

Monday 13 June 2011

cI Repressor Test Assembly, Day 19

     The assemblies that were cultured on 6/10/11 (J23113+B0030+C0051, J23113+B0031+C0051, B0032+C0051 were sent to sequencing. The results showed that only the vector grew, meaning that the assemblies will have to be performed.

     The OR site was placed in front of the cI repressor testing construct assembled on Monday, 6/6/11 as well as in front of a terminator and mCherry reporter.

Assembly Step Part Involved with Assembly Step
Restriction Digest Insert Using SpeI and PstI: OR
Vectors Using XbaI and PstI: K648000+K648001
B0034+mCherry
Ligation OR + K648000+K648001
OR + B0034+mCherry
Transformation/Plating The above ligations were transformed into Escherichia
coli cells and plated onto Kanamycin resistant plates.

Tuesday

cI Repressor Test Assembly, Day 20

     The sensor group re-assembled two ribosome binding sites + cI repressor with a constitutive promoter as the insert.

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

Wednesday

cI Repressor Test Assembly, Day 21

Assembly Step Part Involved in Assembly Step
Restriction Digest Vector using SpeI and PstI: K3+OR
Inserts using XbaI and PstI: B0034+mCherry
K648000+K648001
Vector using EcoRI and XbaI: B0031+C0051
Insert using EcoRI and SpeI: J23113
Ligation OR + B0034+mCherry
OR + K648000+K648001
J23113 + B0031+C0051
Transformation/Plating The above ligations were transformed into
Escherichia coli cells, then plated onto plates
with resistance to the appropriate antibody.

The sensor group also ran an agarose gel electrophoresis test on the assembly from 6/14 and the digests from today in order to see the validity of their work.

Assembly Step Part Involved with Assembly Step
Colony PCR J23113+B0030+C0051 colonies A and B
Agarose Gel
Electrophoresis 		J23113+B0030+C0051 colony A
J23113+B0030+C0051 colony B
K648000+K648001 (Digest)
B0034+mCherry (Digest)
OR (Digest)
B0031+C0051 (Digest)
J23113 (Digest)

Gel Results: The agarose gel contained bands corresponding to the correct length of the J23113+B0030+C0051 assembly for colony B. This colony was allowed to grow overnight in culture.

Thursday

cI Repressor Test Assembly, Day 22

The J23113+B0030+C0051 colony B culture was prepared for sequencing through the Omega Bio-Tek miniprep protocol.

Because many of the assemblies throughout the lab have failed to yield any colonies on the plates, the restriction enzymes were tested. The B0034+C0051 construct was digested with:

EcoRI+SpeI
EcoRI+XbaI
XbaI+PstI
SpeI+PstI
XbaI+SpeI
EcoRI+PstI

These digests were tested using the agarose gel electrophoresis test, but the results were inconclusive.

Back to Notebook

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