Sensor: Week 3 May 30-June 4

From 2011.igem.org

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==Monday==
==Monday==
-
===Testing cI Repressor, Day 6===
+
===cI Repressor Test Assembly, Day 6===
The sensor group ran PCR, transformed, and cultured ribosome binding sites from the registry as well as the cI repressor. The sensor group also prepared for miniprep and sequencing for tomorrow.
The sensor group ran PCR, transformed, and cultured ribosome binding sites from the registry as well as the cI repressor. The sensor group also prepared for miniprep and sequencing for tomorrow.
{|border="1"
{|border="1"
Line 17: Line 17:
==Tuesday==
==Tuesday==
-
===Testing cI Repressor, Day 7===
+
===cI Repressor Test Assembly, Day 7===
The sensor group performed miniprep from the cultures created 5/30/2011. Two RBS and cI repressor constructs were sent to sequencing while assembly was repeated for the other two RBS and the cI repressor since this assembly from last week failed.
The sensor group performed miniprep from the cultures created 5/30/2011. Two RBS and cI repressor constructs were sent to sequencing while assembly was repeated for the other two RBS and the cI repressor since this assembly from last week failed.
{|border="1"
{|border="1"
Line 41: Line 41:
|-
|-
|Transformation/Plating
|Transformation/Plating
-
|colspan="2"|The above ligations were transformed into<br />Escherichia coli cells and plated onto<br />Amp resistant plates
+
|colspan="2"|The above ligations were transformed into<br />Escherichia coli cells and plated onto<br />Amp resistant plates.
|}
|}
 +
 +
==Wednesday==
 +
===cI Repressor Test Assembly, Day 8===
 +
The sensor group placed constitutive promoters in front of the RBS+cI repressor construct and attatched another RBS to the cI repressor.
 +
{|border="1"
 +
!Assembly Step
 +
|colspan="2" align="center"|'''Parts Involved in Assembly Step'''
 +
|-
 +
|rowspan="2"|Restrction Digest
 +
|Inserts using XbaI and PstI:
 +
|J23113<br />J23115<br />C0051
 +
|-
 +
|Vectors using SpeI and PstI:
 +
|B0033<br />B0031+C0051
 +
|-
 +
|Ligation
 +
|colspan="2"|J23113 + B0031+C0051<br />J23105 + B0031+C0051<br />B0033 + C0051
 +
|-
 +
|Transformation/Plating
 +
|colspan="2"|The above ligations were transformed into<br />Escherichia coli cells and plated onto<br />Amp resistant plates.
 +
|}
 +
 +
==Thursday==
 +
===cI Repressor Test Assembly, Day 9===
 +
The sensor group checked the colonies from the assembly performed on 6/1/11. The sensor group continued their attempt to add two RBS in front of the cI repressor. The sensor group also assembled their parts submitted to the registry (the mCherry reporter construct in front of the cI repressor construct).
 +
{|border="1"
 +
!Assembly Step
 +
|colspan="2" align="center"|'''Parts Involved with Assembly Step'''
 +
|-
 +
|Colony PCR
 +
|colspan="2"|J23113+B0031+C0051 (A)<br />J23105+B0031+C0051 (A)<br />B0033+C0051 (A and B)
 +
|-
 +
|rowspan="4"|Restriction Digest
 +
|Insert using EcoRI and SpeI:
 +
|K648000
 +
|-
 +
|Vectors using EcoRI and XbaI:
 +
|K648001<br />K648002
 +
|-
 +
|Insert using XbaI and PstI:
 +
|C0051
 +
|-
 +
|Vectors using SpeI and PstI:
 +
|B0030<br />B0032
 +
|-
 +
|Ligation
 +
|colspan="2"|K648000 + K648001<br />K648000 + K648002<br />B0030 + C0051<br />B0032 + C0051
 +
|-
 +
|Agarose Gel<br />Electrophoresis
 +
|colspan="2"|All colony PCR products from above<br />*''Results: None of the colonies showed the<br />right number of base pairs. Thus, this part<br />of the assembly must be redone.''
 +
|-
 +
|Transformation/Plating
 +
|colspan="2"|The above ligations were transformed into<br />Escherichia coli cells and plated onto<br />Amp resistant plates.
 +
|}
 +
 +
==Friday==
 +
===cI Repressor Test Assembly, Day 10===
 +
The sensor group checked the colonies from the plates created on 6/2/11 through gel electrophoresis.
 +
{|border="1"
 +
!Assembly Step
 +
!Part Involved with Assembly Step
 +
|-
 +
|Colony PCR
 +
|K648000+K648001 (A and B)<br />K648000+K648001 (A and B)<br />B0030+C0051 (A and B)<br />B0032+C0051 (A and B)
 +
|-
 +
|Agarose Gel<br />Electrophoresis
 +
|All colony PCR products from above<br />*''Results: All colonies showed the correct base pair length so all colonies were cultured for further use.''
 +
|}
 +
 +
===Cro and O<sub>R</sub> Assembly via PCR, Day 1===
 +
The sensor group also began assembling the O<sub>R</sub> and Cro parts of the lambda phage system using the designed primers. The parts were first constructed through PCR, then amplified through a second PCR. The first PCR products were labeled with a <span style="color:green">1</span>, and the second PCR products were labelled with a <span style=color:"blue">2</span>.
 +
{|border="1"
 +
!Assembly Step
 +
!Part Involved with<br />Assembly Step
 +
|-
 +
|PCR Assembly
 +
|Cro<br />O<sub>R</sub>
 +
|-
 +
|Agarose Gel<br />Electrophoresis
 +
|Cro <span style="color:green">1</span><br />Cro <span style=color:"blue">2</span><br />O<sub>R</sub> <span style="color:green">1</span><br />O<sub>R</sub> <span style=color:"blue">2</span><br />*''Results: Cro <span style=color:"blue">2</span> and O<sub>R</sub> <span style=color:"blue">2</span><br />proved to contain the<br />correct base pair length.
 +
|}
 +
 +
==Saturday==
 +
===cI Repressor Test Assembly, Day 11===
 +
The sensor group prepared the constructs assembled on 6/2/11 (K648000+K648001, B0030+C0051, B0032+C0051)
 +
for sequencing through miniprep.
 +
 +

Latest revision as of 16:59, 10 June 2011

Contents

Monday

cI Repressor Test Assembly, Day 6

The sensor group ran PCR, transformed, and cultured ribosome binding sites from the registry as well as the cI repressor. The sensor group also prepared for miniprep and sequencing for tomorrow.

Assembly step Parts Ivolved with Assembly Step
PCR C0051 from kit
Transform/Culture B0030
B0032
C0051 from kit
Colony Growth (for sequencing) B0031+C0051
B0033+C0051

Tuesday

cI Repressor Test Assembly, Day 7

The sensor group performed miniprep from the cultures created 5/30/2011. Two RBS and cI repressor constructs were sent to sequencing while assembly was repeated for the other two RBS and the cI repressor since this assembly from last week failed.

Assembly Step Parts Involved with Assembly Step
Miniprep For Sequencing: B0031+C0051
B0033+C0051
For Assembly: C0051
B0030
B0032
Restriction Digest Insert using XbaI and PstI: C0051
Vector using SpeI and PstI B0030
B0032
Ligation B0030+C0051
B0032+C0051
Transformation/Plating The above ligations were transformed into
Escherichia coli cells and plated onto
Amp resistant plates.

Wednesday

cI Repressor Test Assembly, Day 8

The sensor group placed constitutive promoters in front of the RBS+cI repressor construct and attatched another RBS to the cI repressor.

Assembly Step Parts Involved in Assembly Step
Restrction Digest Inserts using XbaI and PstI: J23113
J23115
C0051
Vectors using SpeI and PstI: B0033
B0031+C0051
Ligation J23113 + B0031+C0051
J23105 + B0031+C0051
B0033 + C0051
Transformation/Plating The above ligations were transformed into
Escherichia coli cells and plated onto
Amp resistant plates.

Thursday

cI Repressor Test Assembly, Day 9

The sensor group checked the colonies from the assembly performed on 6/1/11. The sensor group continued their attempt to add two RBS in front of the cI repressor. The sensor group also assembled their parts submitted to the registry (the mCherry reporter construct in front of the cI repressor construct).

Assembly Step Parts Involved with Assembly Step
Colony PCR J23113+B0031+C0051 (A)
J23105+B0031+C0051 (A)
B0033+C0051 (A and B)
Restriction Digest Insert using EcoRI and SpeI: K648000
Vectors using EcoRI and XbaI: K648001
K648002
Insert using XbaI and PstI: C0051
Vectors using SpeI and PstI: B0030
B0032
Ligation K648000 + K648001
K648000 + K648002
B0030 + C0051
B0032 + C0051
Agarose Gel
Electrophoresis 		All colony PCR products from above
*Results: None of the colonies showed the
right number of base pairs. Thus, this part
of the assembly must be redone.
Transformation/Plating The above ligations were transformed into
Escherichia coli cells and plated onto
Amp resistant plates.

Friday

cI Repressor Test Assembly, Day 10

The sensor group checked the colonies from the plates created on 6/2/11 through gel electrophoresis.

Assembly Step Part Involved with Assembly Step
Colony PCR K648000+K648001 (A and B)
K648000+K648001 (A and B)
B0030+C0051 (A and B)
B0032+C0051 (A and B)
Agarose Gel
Electrophoresis 		All colony PCR products from above
*Results: All colonies showed the correct base pair length so all colonies were cultured for further use.

Cro and OR Assembly via PCR, Day 1

The sensor group also began assembling the OR and Cro parts of the lambda phage system using the designed primers. The parts were first constructed through PCR, then amplified through a second PCR. The first PCR products were labeled with a 1, and the second PCR products were labelled with a 2.

Assembly Step Part Involved with
Assembly Step
PCR Assembly Cro
OR
Agarose Gel
Electrophoresis 		Cro 1
Cro 2
OR 1
OR 2
*Results: Cro 2 and OR 2
proved to contain the
correct base pair length.

Saturday

cI Repressor Test Assembly, Day 11

The sensor group prepared the constructs assembled on 6/2/11 (K648000+K648001, B0030+C0051, B0032+C0051) for sequencing through miniprep.




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