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Sensor: Week 4 June 6-11
From 2011.igem.org
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 2
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
cI Repressor Test Assembly, Day 16
The sensor group checked to see if the assemblies from 6/9 worked through agarose gel electrophoresis.
| Assembly Step | Part Involved with Assembly Step |
|---|---|
| Colony PCR | B0032+C0051 J23113+B0031+C0051 |
| Agarose Gel Electrophoresis | B0032+C0051 J23113+B0031+C0051 |
| Culture (for sequencing) | J23113+B0030+C0051 J23113+B0031+C0051 B0032+C0051 |
*Results: The gel electrophoresis showed that the B0032+C0051 construct contained the correct number of base pairs. However, no bands showed for the J23113+B0031+C0051 construct. Both constructs were prepared for sequencing to further determine the legitimacy of the assembly.
Cro and OR Insert into K3 Vector, Day 3
K3 + OR colony A
K3 + OR colony B
K3 + Cro colony A
K3 + Cro colony B
were all sent to sequencing. The sequence of the OR+K3 colonies showed that this assembly worked. The Cro+K3 colony A had one base pair change, and Cro+K3 colony B had a base pair deletion. Thus, freezer stock of OR+K3 will be prepared and the Cro+K3 assembly will be redone.
Saturday
cI Repressor Test Assembly, Day 17
J23113+B0030+C0051
J23113+B0031+C0051
B0032+C0051
were miniprepped in order to send to sequencing on Monday, 6/13/11.
The K648000+K648001 insert was amplified through PCR, but did not show up on an agarose gel electrophoresis test. OneTaq polymerase was used for this PCR, which probably went wrong and should be redone.
