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Sensor: Week 4 June 6-11
From 2011.igem.org
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===Cro and O<sub>R</sub> Insert into C3 Vector, Day 2=== | ===Cro and O<sub>R</sub> 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 O<sub>R</sub> must be inserted into a different vector.''' | All of the colonies on the plates were purple, meaning that the insert never inserted into the vector. Thus, '''Cro and O<sub>R</sub> must be inserted into a different vector.''' | ||
| + | |||
==Wednesday== | ==Wednesday== | ||
===cI Repressor Test Assembly, Day 14=== | ===cI Repressor Test Assembly, Day 14=== | ||
| - | The B0034+C0051 construct was sent to sequencing. Freezer stocks were made from the cultures made 6/7 (listed above). | + | 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 O<sub>R</sub> Insert into K3 Vector, Day 1=== | ===Cro and O<sub>R</sub> Insert into K3 Vector, Day 1=== | ||
Since the C3 vector did not accept the Cro and O<sub>R</sub> inserts, the sensor group attempted to insert Cro and O<sub>R</sub> into the K3 vector. | Since the C3 vector did not accept the Cro and O<sub>R</sub> inserts, the sensor group attempted to insert Cro and O<sub>R</sub> into the K3 vector. | ||
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|} | |} | ||
| + | ==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. | ||
| + | {|border="1" | ||
| + | !Assembly Step | ||
| + | |colspan="2" align="center"|'''Part Involved with Assembly Step''' | ||
| + | |- | ||
| + | |rowspan="4"|Restriction Digest | ||
| + | |Insert using EcoRI and SpeI: | ||
| + | |J23113<br />B0033 | ||
| + | |- | ||
| + | |Vector using EcoRI and XbaI: | ||
| + | |B0030+C0051<br />B0031+C0051<br />C0051 | ||
| + | |- | ||
| + | |Insert using XbaI and PstI: | ||
| + | |C0051 | ||
| + | |- | ||
| + | |Vector using SpeI and PstI: | ||
| + | |B0032 | ||
| + | |- | ||
| + | |Ligation | ||
| + | |colspan="2"|J23113 + B0030+C0051<br />J23113 + B0031+C0051<br />B0033 + C0051<br />B0032 + C0051 | ||
| + | |- | ||
| + | |Transformation/Plating | ||
| + | |colspan="2" |The above ligations were transformed into<br />Escherichia coli cells and plated onto<br />ampicillin resistant plates. | ||
| + | |} | ||
| + | ===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 | ||
| + | were all cultured in order to have the constructs sequenced. | ||
| + | ==Friday== | ||
| + | === | ||
[[Team:Penn_State/Notebook| Back to Notebook]] | [[Team:Penn_State/Notebook| Back to Notebook]] | ||
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.
