Sensor: Week 8 July 5-8
From 2011.igem.org
(One intermediate revision not shown) | |||
Line 1: | Line 1: | ||
==Tuesday, July 5== | ==Tuesday, July 5== | ||
+ | ===cI Repressor Test Assembly, Day 27=== | ||
+ | ''Sequencing Results:'' The following constructs were sent to sequencing to verify their DNA sequences: | ||
+ | {|border="1" | ||
+ | !Construct | ||
+ | !Sequencing<br />Result | ||
+ | |- | ||
+ | |J23113 + B0034 + C0051 | ||
+ | |align="center"|Good | ||
+ | |- | ||
+ | |KOR + K648000 | ||
+ | |align="center"|Good | ||
+ | |} | ||
+ | |||
===Clone O<sub>R</sub> and CRO=== | ===Clone O<sub>R</sub> and CRO=== | ||
{|border="1" | {|border="1" | ||
Line 13: | Line 26: | ||
|- | |- | ||
|Ligation | |Ligation | ||
- | |colspan="2"|K3 + OR<br />K3 + CRO | + | |colspan="2" align="center"|K3 + OR<br />K3 + CRO |
|- | |- | ||
|Transformation/Plating | |Transformation/Plating | ||
Line 19: | Line 32: | ||
|} | |} | ||
- | ===cI Repressor Test Assembly, Day | + | ==Wednesday, July 6== |
- | + | ===cI Repressor Test Assembly, Day 28=== | |
+ | The sensor group attempted to place two ribosome binding sites behind the cI repressor. | ||
{|border="1" | {|border="1" | ||
- | ! | + | !Protocol |
- | + | |colspan="2" align="center"|'''Part Involved with Protocol''' | |
|- | |- | ||
- | | | + | |Insert PCR |
- | |align="center"| | + | |colspan="2" align="center"|B0033<br />B0032 |
|- | |- | ||
- | | | + | |rowspan="2"|Restriction Digest |
- | |align="center"| | + | |Inserts using EcoRI and SpeI: |
+ | |B0032<br />B0033 | ||
+ | |- | ||
+ | |Vector using EcoRI and XbaI: | ||
+ | |C0051 | ||
+ | |- | ||
+ | |Ligation | ||
+ | |colspan="2" align="center"|C0051 + B0032<br />C0051 + B0033 | ||
+ | |- | ||
+ | |Transformation/Plating | ||
+ | |colspan="2"|The above ligations were transformed into<br />Escherichia coli cells and plated onto<br />ampicillin resistant plates. | ||
|} | |} | ||
+ | ===Clone O<sub>R</sub> and CRO, Take 2=== | ||
+ | No colonies grew on the kanamycin resistant plates from 7/5, so the cloning was attempted again. | ||
+ | {|border="1" | ||
+ | !Protocol | ||
+ | |colspan="2" align="center"|'''Part Involved with Protocol''' | ||
+ | |- | ||
+ | |rowspan="2"|Restriction Digest | ||
+ | |Inserts using XbaI and PstI: | ||
+ | |O<sub>R</sub><br />CRO | ||
+ | |- | ||
+ | |Vector using XbaI and PstI: | ||
+ | |K3 | ||
+ | |- | ||
+ | |Ligation | ||
+ | |colspan="2" align="center"|K3 + OR<br />K3 + CRO | ||
+ | |- | ||
+ | |Transformation/Plating | ||
+ | |colspan="2"|The above ligations were transformed into<br />Escherichia coli cells and plated onto<br />kanamycin resistant plates. | ||
+ | |} | ||
+ | ==Thursday, July 7== | ||
+ | ===cI Repressor Test Assembly, Day 29=== | ||
+ | The sensor group verified their assemblies from 7/6 through agarose gel electrophoresis. The C0051+B0033 looked good, but the C0051+B0032 did not. | ||
+ | ==Friday, July 8== | ||
+ | The C0051+B0033 and K3+CRO constructs were sent to the sequencing center. The C0051+B0033 DNA sequence came back positive, but the K3+CRO did not, so this cloning attempt must be done again. | ||
+ | |||
+ | Since the sequencing results came back positive, freezer stocks were made containing J23113+B0034+C0051 and KO<sub>R</sub>+K648000. | ||
+ | |||
+ | Colonies of the following constructs were grown up: | ||
+ | <blockquote>KCRO</blockquote> | ||
+ | <blockquote>KO<sub>R</sub></blockquote> | ||
+ | <blockquote>B0032+C0051</blockquote> | ||
+ | <blockquote>B0033+C0051</blockquote> | ||
+ | |||
+ | ===cI Repressor Test Assembly, Day 30=== | ||
+ | {|border="1" | ||
+ | !Protocol | ||
+ | |colspan="2" align="center"|'''Part Involved with Protocol''' | ||
+ | |- | ||
+ | |Insert PCR | ||
+ | |colspan="2" align="center"|J23113 + B0031 + C0051<br />J23113 + B0034 + C0051<br />J2313 + B0030 + C0051 | ||
+ | |- | ||
+ | |rowspan="2"|Restriction Digest | ||
+ | |Inserts using XbaI and PstI: | ||
+ | |J23113 + B0031 + C0051<br />J23113 + B0034 + C0051<br />J2313 + B0030 + C0051 | ||
+ | |- | ||
+ | |Vector using SpeI and PstI: | ||
+ | |O<sub>R</sub> | ||
+ | |- | ||
+ | |Ligation | ||
+ | |colspan="2"|OR + K648000 + J23113 + B0031 + C0051<br />OR + K648000 + J23113 + B0034 + C0051<br />OR + K648000 + J23113 + B0030 + C0051 | ||
+ | |- | ||
+ | |Transformation/Plating | ||
+ | |colspan="2"|The above ligations were transformed into Escherichia<br />coli cells and plated onto kanamycin resistant plates. | ||
+ | |} | ||
[[Team:Penn_State/Notebook| Back to Notebook]] | [[Team:Penn_State/Notebook| Back to Notebook]] |
Latest revision as of 18:27, 13 July 2011
Contents |
Tuesday, July 5
cI Repressor Test Assembly, Day 27
Sequencing Results: The following constructs were sent to sequencing to verify their DNA sequences:
Construct | Sequencing Result |
---|---|
J23113 + B0034 + C0051 | Good |
KOR + K648000 | Good |
Clone OR and CRO
Protocol | Part Involved with Protocol | |
---|---|---|
Restriction Digest | Inserts using XbaI and PstI: | OR CRO |
Vector using XbaI and PstI: | K3 | |
Ligation | K3 + OR K3 + CRO | |
Transformation/Plating | The above ligations were transformed into Escherichia coli cells and plated onto kanamycin resistant plates. |
Wednesday, July 6
cI Repressor Test Assembly, Day 28
The sensor group attempted to place two ribosome binding sites behind the cI repressor.
Protocol | Part Involved with Protocol | |
---|---|---|
Insert PCR | B0033 B0032 | |
Restriction Digest | Inserts using EcoRI and SpeI: | B0032 B0033 |
Vector using EcoRI and XbaI: | C0051 | |
Ligation | C0051 + B0032 C0051 + B0033 | |
Transformation/Plating | The above ligations were transformed into Escherichia coli cells and plated onto ampicillin resistant plates. |
Clone OR and CRO, Take 2
No colonies grew on the kanamycin resistant plates from 7/5, so the cloning was attempted again.
Protocol | Part Involved with Protocol | |
---|---|---|
Restriction Digest | Inserts using XbaI and PstI: | OR CRO |
Vector using XbaI and PstI: | K3 | |
Ligation | K3 + OR K3 + CRO | |
Transformation/Plating | The above ligations were transformed into Escherichia coli cells and plated onto kanamycin resistant plates. |
Thursday, July 7
cI Repressor Test Assembly, Day 29
The sensor group verified their assemblies from 7/6 through agarose gel electrophoresis. The C0051+B0033 looked good, but the C0051+B0032 did not.
Friday, July 8
The C0051+B0033 and K3+CRO constructs were sent to the sequencing center. The C0051+B0033 DNA sequence came back positive, but the K3+CRO did not, so this cloning attempt must be done again.
Since the sequencing results came back positive, freezer stocks were made containing J23113+B0034+C0051 and KOR+K648000.
Colonies of the following constructs were grown up:
KCRO
KOR
B0032+C0051
B0033+C0051
cI Repressor Test Assembly, Day 30
Protocol | Part Involved with Protocol | |
---|---|---|
Insert PCR | J23113 + B0031 + C0051 J23113 + B0034 + C0051 J2313 + B0030 + C0051 | |
Restriction Digest | Inserts using XbaI and PstI: | J23113 + B0031 + C0051 J23113 + B0034 + C0051 J2313 + B0030 + C0051 |
Vector using SpeI and PstI: | OR | |
Ligation | OR + K648000 + J23113 + B0031 + C0051 OR + K648000 + J23113 + B0034 + C0051 OR + K648000 + J23113 + B0030 + C0051 | |
Transformation/Plating | The above ligations were transformed into Escherichia coli cells and plated onto kanamycin resistant plates. |