Reporter: Week 10 July 17-23

From 2011.igem.org

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==Sunday, July 17==
==Sunday, July 17==
 +
No colonies grew for tev cleavage site+GFP, and only one colony grew for cI cleavage site+GFP. This colony was amplified through colony PCR (extension time=1:20). The PCR products were run on an agarose gel, which yielded a band that corresponded to around 1000 bp, which was expected. The colony was grown up in culture overnight.
 +
==Monday, July 18==
 +
The tev mutagenesis and cI cleavage site+GFP plasmids were sent to the sequencing center. The sequencing results showed that the cI cleavage site+GFP worked, but the tev mutagenesis did not.
 +
Since we have had trouble inserting the cleavage sites in front of the GFP gene, the GFP was inserted behind the cleavage sites.
 +
{|border="1"
 +
!Protocol
 +
|colspan="2" align="center"|'''Part Involved with Protocol'''
 +
|-
 +
|Insert PCR (extension time=1:15)
 +
|colspan="2"|GFP
 +
|-
 +
|rowspan="2"|Restriction Digest
 +
|Insert using NgoMIV and PstI (buffer 1)
 +
|GFP
 +
|-
 +
|Vector using AgeI and PstI (buffer 1):
 +
|tev cleavage site
 +
|-
 +
|Ligation
 +
|colspan="2"|tev cleavage site+GFP
 +
|-
 +
|Transformation/Plating
 +
|colspan="2"|The above ligation was transformed into Escherichia<br />coli cells and plated on kanamycin resistant plates.
 +
|}
 +
 +
The promoter and RBS construct (J23100+B0034) was placed in front of GFP+tev cleavage site, XylE+small linker and XylE+Imp linker following the procedure performed on Thursday, July 7.
 +
 +
The GusA and LacZ&alpha; genes were cloned into K3 using the 10AA linker+K3 as the vector.
 +
 +
==Tuesday, July 19==
 +
The promoter and RBS colonies that started growing on Monday, July 18 were amplified through colony PCR (extension time=1:15). The PCR products were run on an agarose gel, which yielded bands that corresponded to the correct number of base pairs. These colonies were grown up in culture. The J23100+B0024+XylE+10AA linker and J23100+B0024+GFP+cI cleavage site cultures were frozen as stocks.
 +
 +
The GusA and LacZ&alpha; genes were cloned into K3 following the same procedure performed on Monday, July 18.
 +
 +
The linkers were placed in front of XylE.
 +
{|border="1"
 +
!Protocol
 +
|colspan="2" align="center"|'''Part Involved with Protocol
 +
|-
 +
|rowspan="2"|Restriction Digest
 +
|Insert using NgoMIV and PstI (buffer 1):
 +
|XylE
 +
|-
 +
|Vector using AgeI and PstI (buffer 1):
 +
|10 AA linker<br />Imp linker<br />small linker
 +
|-
 +
|Ligation
 +
|colspan="2"|10AA linker+XylE<br />Imp linker+XylE<br />small linker+XylE
 +
|-
 +
|Transformation/Plating
 +
|colspan="2"|The above ligations were transformed into<br />Escherichia coli cells and plated on kanamycin<br />resistant plates.
 +
|}
 +
 +
''Results:'' The 10 AA liker+XylE worked, but the Imp linker+XylE and small liker+XylE must be repeated.
 +
 +
==Thursday, July 21==
 +
The promoter and RBS assembly from Wednesday, July 20, was verified. Four colonies were amplified through colony PCR. The PCR products were run on an agarose gel, which showed bands that corresponded to the expected sizes. The colonies were grown up in culture.
 +
 +
The sequencing results for the GusA and LacZ&alpha; cloning attempts from Monday, July 18 and Tuesday, July 19 showed that GusA was missing the NgoMIV and AgeI sites. Also, both LacZ&alpha; and GusA were cut in the wrong place, resulting in the appearance of only part of the correct sequence. Nexte time, separate insert PCRs will be run with higher annealing temperatures.
 +
 +
==Friday, July 22==
 +
The cleavage sites+GFP constructs were placed behind the J23100+B0034+XylE+linker constructs.
 +
{|border="1"
 +
!Protocol
 +
|colspan="2" align="center"|'''Part Involved with Protocol'''
 +
|-
 +
|Insert PCR<br/>(extension time=1:20)
 +
|colspan="2"|tev cleavage site+GFP<br />cI cleavage site+GFP
 +
|-
 +
|rowspan="2"|Restriction Digest
 +
|Inserts using NgoMIV and PstI (buffer 1):
 +
|tev cleavage site+GFP<br />cI cleavage site+GFP
 +
|-
 +
|Vectors using AgeI and PstI (buffer 1):
 +
|J23100+B0034+XylE+small linker<br />J23100+B0034+XylE+Imp linker<br />J23100+B0034+XylE+10AA linker
 +
|-
 +
|Ligation
 +
|colspan="2"|J23100+B0034+XylE+small linker+tev cleavage site+GFP<br />J23100+B0034+XylE+small linker+cI cleavage site+GFP<br />J23100+B0034+XylE+Imp linker+tev cleavage site+GFP<br />J23100+B0034+XylE+Imp linker+cI cleavage site+GFP<br />J23100+B0034+Xyle+10AA linker+tev cleavage site+GFP<br />J23100+B0034+XylE+10AA linker+cI cleavage site+GFP
 +
|-
 +
|Transformation/Plating
 +
|colspan="2"|The above ligations were transformed into Escherichia coli cells and<br />plated onto kanamycin resistant plates.
 +
|}
 +
 +
==Saturday, July 23==
 +
Clone LacZ&alpha; into K3
 +
 +
Miniprep tev protease and 10AA linker for sequencing
 +
Results: the 10AA linker was cloned correctly, but the tev protease failed
 +
 +
Insert PCR of mutated XylE
[[Team:Penn_State/Notebook| Back to Notebook]]
[[Team:Penn_State/Notebook| Back to Notebook]]

Latest revision as of 06:47, 28 September 2011

Contents

Sunday, July 17

No colonies grew for tev cleavage site+GFP, and only one colony grew for cI cleavage site+GFP. This colony was amplified through colony PCR (extension time=1:20). The PCR products were run on an agarose gel, which yielded a band that corresponded to around 1000 bp, which was expected. The colony was grown up in culture overnight.

Monday, July 18

The tev mutagenesis and cI cleavage site+GFP plasmids were sent to the sequencing center. The sequencing results showed that the cI cleavage site+GFP worked, but the tev mutagenesis did not.

Since we have had trouble inserting the cleavage sites in front of the GFP gene, the GFP was inserted behind the cleavage sites.

Protocol Part Involved with Protocol
Insert PCR (extension time=1:15) GFP
Restriction Digest Insert using NgoMIV and PstI (buffer 1) GFP
Vector using AgeI and PstI (buffer 1): tev cleavage site
Ligation tev cleavage site+GFP
Transformation/Plating The above ligation was transformed into Escherichia
coli cells and plated on kanamycin resistant plates.

The promoter and RBS construct (J23100+B0034) was placed in front of GFP+tev cleavage site, XylE+small linker and XylE+Imp linker following the procedure performed on Thursday, July 7.

The GusA and LacZα genes were cloned into K3 using the 10AA linker+K3 as the vector.

Tuesday, July 19

The promoter and RBS colonies that started growing on Monday, July 18 were amplified through colony PCR (extension time=1:15). The PCR products were run on an agarose gel, which yielded bands that corresponded to the correct number of base pairs. These colonies were grown up in culture. The J23100+B0024+XylE+10AA linker and J23100+B0024+GFP+cI cleavage site cultures were frozen as stocks.

The GusA and LacZα genes were cloned into K3 following the same procedure performed on Monday, July 18.

The linkers were placed in front of XylE.

Protocol Part Involved with Protocol
Restriction Digest Insert using NgoMIV and PstI (buffer 1): XylE
Vector using AgeI and PstI (buffer 1): 10 AA linker
Imp linker
small linker
Ligation 10AA linker+XylE
Imp linker+XylE
small linker+XylE
Transformation/Plating The above ligations were transformed into
Escherichia coli cells and plated on kanamycin
resistant plates.

Results: The 10 AA liker+XylE worked, but the Imp linker+XylE and small liker+XylE must be repeated.

Thursday, July 21

The promoter and RBS assembly from Wednesday, July 20, was verified. Four colonies were amplified through colony PCR. The PCR products were run on an agarose gel, which showed bands that corresponded to the expected sizes. The colonies were grown up in culture.

The sequencing results for the GusA and LacZα cloning attempts from Monday, July 18 and Tuesday, July 19 showed that GusA was missing the NgoMIV and AgeI sites. Also, both LacZα and GusA were cut in the wrong place, resulting in the appearance of only part of the correct sequence. Nexte time, separate insert PCRs will be run with higher annealing temperatures.

Friday, July 22

The cleavage sites+GFP constructs were placed behind the J23100+B0034+XylE+linker constructs.

Protocol Part Involved with Protocol
Insert PCR
(extension time=1:20) 		tev cleavage site+GFP
cI cleavage site+GFP
Restriction Digest Inserts using NgoMIV and PstI (buffer 1): tev cleavage site+GFP
cI cleavage site+GFP
Vectors using AgeI and PstI (buffer 1): J23100+B0034+XylE+small linker
J23100+B0034+XylE+Imp linker
J23100+B0034+XylE+10AA linker
Ligation J23100+B0034+XylE+small linker+tev cleavage site+GFP
J23100+B0034+XylE+small linker+cI cleavage site+GFP
J23100+B0034+XylE+Imp linker+tev cleavage site+GFP
J23100+B0034+XylE+Imp linker+cI cleavage site+GFP
J23100+B0034+Xyle+10AA linker+tev cleavage site+GFP
J23100+B0034+XylE+10AA linker+cI cleavage site+GFP
Transformation/Plating The above ligations were transformed into Escherichia coli cells and
plated onto kanamycin resistant plates.

Saturday, July 23

Clone LacZα into K3

Miniprep tev protease and 10AA linker for sequencing Results: the 10AA linker was cloned correctly, but the tev protease failed

Insert PCR of mutated XylE

Back to Notebook

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