Sensor: Week 7 June 28-July 1

From 2011.igem.org

(Difference between revisions)
 
(3 intermediate revisions not shown)
Line 21: Line 21:
|-
|-
|Ligation
|Ligation
-
|colspan="2"|B0034 + K3 + Cro<br />KOr + K648000 + K648001<br />KOr + 34mcherry<br />B0032 + C0051
+
|colspan="2"|B0034 + K3 + Cro<br />KO<sub>R</sub> + K648000 + K648001<br />KO<sub>R</sub> + 34mcherry<br />B0032 + C0051
|-
|-
|Transformation/Plating
|Transformation/Plating
-
|colspan="2"|The above ligations were transformed into<br />Escherichia coli cells and plated on kanamycin<br />resistant plates.
+
|colspan="2"|The above ligations were transformed into<br />Escherichia coli cells and plated on kanamycin<br /> and ampicillin resistant plates.
|}
|}
&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;The sensor group also transformed K3+Cro and J23113+B0031+C0051 in order to make stocks. The B0034+C0051 was grown up from stock.
&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;The sensor group also transformed K3+Cro and J23113+B0031+C0051 in order to make stocks. The B0034+C0051 was grown up from stock.
 +
==Wednesday, June 29==
 +
===cI Repressor Test Assembly, Day 24===
 +
The sensor group verified their assemblies from 6/28. Colonies containing KO<sub>R</sub> + 34mcherry, B0034 + K3 + Cro, KOr + K648000+K648001 were amplified through colony PCR. Agarose gel electrophoresis showed that these assemblies seemed to work. The colonies were grown up in culture so the plasmids can be extracted and sent to the sequencing center tomorrow morning.
 +
The B0034 and B0034+C0051 plasmids were extracted using the Omega-Biotek miniprep protocol. These plasmids will be used at a later date.
 +
 +
==Thursday, June 30==
 +
===cI Repressor Test Assembly, Day 25===
 +
The following constructs made on 6/28 were sent to sequencing.
 +
<blockquote>KOr + 34mcherry</blockquote>
 +
<blockquote>B0034 + K3 + Cro</blockquote>
 +
<blockquote>KOr + K648000 + K648001</blockquote>
 +
The results showed that these colonies were just vector background. As a result, these assemblies must be done again.
 +
 +
The sensor group also assembled a constitutive promoter to two RBS+cI repressor part. Also, the KO<sub>R</sub> part was placed in front of the mCherry+terminator part.
 +
{|border="1"
 +
!Protocol
 +
|align="center" colspan="2" |Part Involved with Protocol'''
 +
|-
 +
|rowspan="4"|Restriction Digest
 +
|Inserts using EcoRI and SpeI:
 +
|J23113<br />B0032<br />B0033
 +
|-
 +
|Insert using XbaI and PstI:
 +
|K648000
 +
|-
 +
|Vectors using EcoRI and XbaI:
 +
|B0034 + C0051<br />C0051
 +
|-
 +
|Vector using SpeI and PstI:
 +
|KO<sub>R</sub>
 +
|-
 +
|Ligation
 +
|colspan="2"|J23113 + B0032 + C0051<br />J23113 + B0033 + C0051<br />KOr + K648000
 +
|-
 +
|Transformation/Plating
 +
|colspan="2"|The above ligations were transformed into<br />Escherichia coli cells and plated on<br />kanamycin and ampicillin resistant plates.
 +
|}
 +
 +
The cI repressor (C0051) plasmid was transformed into Escherichia coli cells from the registry kit plate.
 +
 +
The following constructs were sent to sequencing:
 +
{|border="1"
 +
!Construct
 +
!Sequencing<br />Result
 +
|-
 +
|J23113 + B0032 + C0051
 +
|good
 +
|-
 +
|J23113 + B0033 + C0051
 +
|bad
 +
|-
 +
|KOr + K648000
 +
|good
 +
|}
 +
 +
==Friday, July 1==
 +
===cI Repressor Test Assembly, Day 26===
 +
The sensor group tried attaching the cI repressor to two different RBS.
 +
 +
{|border="1"
 +
!Protocol
 +
|colspan="2" align="center"|'''Part Involved with Protocol'''
 +
|-
 +
|rowspan="2"|Restriction Digest
 +
|Inserts with EcoRI and SpeI:
 +
|B0032<br />B0033
 +
|-
 +
|Vector with EcoRI and XbaI:
 +
|C0051
 +
|-
 +
|Ligation
 +
|colspan="2"|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.
 +
|}
[[Team:Penn_State/Notebook| Back to Notebook]]
[[Team:Penn_State/Notebook| Back to Notebook]]

Latest revision as of 17:19, 13 July 2011

Contents

Tuesday, June 28

cI Repressor Test Assembly, Day 23

     The sensor group continued their assembly of their testing construct. They placed an RBS in the K3 vector with Cro, the OR in front of the mCherry reporter, terminator, cI repressor and J23113 promoter as well as in front of the 34mCherry. Finally, the sensor group attempted (again) to put the B0032 RBS in front of the cI repressor.

Protocol Part Involved with Protocol
Restriction Digest Inserts using XbaI and PstI: 34mCherry
K648000+K648001
Inserts using EcoRI and SpeI: B0032
B0034
Vectors using EcoRI and XbaI: C0051
K3+Cro
Vector using SpeI and PstI: K3+OR
Ligation B0034 + K3 + Cro
KOR + K648000 + K648001
KOR + 34mcherry
B0032 + C0051
Transformation/Plating The above ligations were transformed into
Escherichia coli cells and plated on kanamycin
and ampicillin resistant plates.

     The sensor group also transformed K3+Cro and J23113+B0031+C0051 in order to make stocks. The B0034+C0051 was grown up from stock.

Wednesday, June 29

cI Repressor Test Assembly, Day 24

The sensor group verified their assemblies from 6/28. Colonies containing KOR + 34mcherry, B0034 + K3 + Cro, KOr + K648000+K648001 were amplified through colony PCR. Agarose gel electrophoresis showed that these assemblies seemed to work. The colonies were grown up in culture so the plasmids can be extracted and sent to the sequencing center tomorrow morning.

The B0034 and B0034+C0051 plasmids were extracted using the Omega-Biotek miniprep protocol. These plasmids will be used at a later date.

Thursday, June 30

cI Repressor Test Assembly, Day 25

The following constructs made on 6/28 were sent to sequencing.

KOr + 34mcherry
B0034 + K3 + Cro
KOr + K648000 + K648001

The results showed that these colonies were just vector background. As a result, these assemblies must be done again.

The sensor group also assembled a constitutive promoter to two RBS+cI repressor part. Also, the KOR part was placed in front of the mCherry+terminator part.

Protocol Part Involved with Protocol
Restriction Digest Inserts using EcoRI and SpeI: J23113
B0032
B0033
Insert using XbaI and PstI: K648000
Vectors using EcoRI and XbaI: B0034 + C0051
C0051
Vector using SpeI and PstI: KOR
Ligation J23113 + B0032 + C0051
J23113 + B0033 + C0051
KOr + K648000
Transformation/Plating The above ligations were transformed into
Escherichia coli cells and plated on
kanamycin and ampicillin resistant plates.

The cI repressor (C0051) plasmid was transformed into Escherichia coli cells from the registry kit plate.

The following constructs were sent to sequencing:

Construct Sequencing
Result
J23113 + B0032 + C0051 good
J23113 + B0033 + C0051 bad
KOr + K648000 good

Friday, July 1

cI Repressor Test Assembly, Day 26

The sensor group tried attaching the cI repressor to two different RBS.

Protocol Part Involved with Protocol
Restriction Digest Inserts with EcoRI and SpeI: B0032
B0033
Vector with 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.


Back to Notebook

Retrieved from "http://2011.igem.org/Sensor:_Week_7_June_28-July_1"