Team:UAB-Barcelona/CS1

From 2009.igem.org

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After designing the biobricks we proceeded to transform those we needed, using a method of chemical transformation and with competent cells prepared with a new improved protocol.
After designing the biobricks we proceeded to transform those we needed, using a method of chemical transformation and with competent cells prepared with a new improved protocol.
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In the first transformation that we realize, we had a low efficiency. We only obtained colonies of the biobricks with resistance to kanamycin. Because of it we prepared plates with lower Ampicillin's concentration (50 g/ml).  
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In the first transformation that we did, we had a low efficiency. We only obtained colonies of the biobricks with resistance to kanamycin. Because of it we prepared plates with lower Ampicillin's concentration (50 g/ml).  
We increased the efficiency of transformation adding one more step to our protocol: 1 min in ice after the 45 s in 42ºC.  
We increased the efficiency of transformation adding one more step to our protocol: 1 min in ice after the 45 s in 42ºC.  
We extracted the plasmidic DNA (minipreps) with the kit Wizard Plus SV Minipreps DNA Purification System (Promega) <html><a href="https://static.igem.org/mediawiki/2009/1/1e/Protocols.pdf">(PROTOCOL)</a></html>.
We extracted the plasmidic DNA (minipreps) with the kit Wizard Plus SV Minipreps DNA Purification System (Promega) <html><a href="https://static.igem.org/mediawiki/2009/1/1e/Protocols.pdf">(PROTOCOL)</a></html>.
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==Cloning strategy==
==Cloning strategy==
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''RBS-RFP-T-T''
''RBS-RFP-T-T''
P3 13K
P3 13K
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|}
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|}
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==Biobrick digestion==
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https://static.igem.org/mediawiki/2009/7/77/D1.jpg
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'''Agarose gel digestion purification.'''
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From left side to right: BBa_J04450 digested and no digested; BBa_I13521 digested y no digested; lambda-DNA hinIII; nirk ; nsr; phoa
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https://static.igem.org/mediawiki/2009/5/53/D2.jpg
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'''Agarose gel digestion purification.'''
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 +
From left side to right: BBa_B0014 digestedand no digested; lambda-DNA hinIII; BBa_J61127 digested and no digested; lambda-DNA hinIII ; nirk ; BBa_B0040 digested and no digested.
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https://static.igem.org/mediawiki/2009/c/ce/D3.jpg
 +
 +
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'''Agarose gel digestion purification.'''
 +
 +
From left side to right: BBa_E0840 digested and no digested; lambda-DNA hinIII; BBa_I13504 digested and no digested; lambda-DNA hinIII; nirk ; BBa_K093005 digested and no digested.

Latest revision as of 03:36, 22 October 2009

Encap raro.jpg Documento sin título

Contents

Transformation with biobricks

After designing the biobricks we proceeded to transform those we needed, using a method of chemical transformation and with competent cells prepared with a new improved protocol.

In the first transformation that we did, we had a low efficiency. We only obtained colonies of the biobricks with resistance to kanamycin. Because of it we prepared plates with lower Ampicillin's concentration (50 g/ml).

We increased the efficiency of transformation adding one more step to our protocol: 1 min in ice after the 45 s in 42ºC.

We extracted the plasmidic DNA (minipreps) with the kit Wizard Plus SV Minipreps DNA Purification System (Promega) (PROTOCOL).

Cloning strategy

1st round

phoa (E,S) + backbone BBa_K093005 (E,S) // Biobrick 1

nirk (E,S) + backbone BBa_K093005 (E,S) // Biobrick 2

nsr (X,P) + backbone BBa_K093005 (E,S) // Biobrick 3


phoa (E,S) + RBS-LacI BBa_J24679 (E,X) // Biobrick 4

pLacI-RBS-RFP-T-T BBa_J04450 (E,S) + linker BBa_B0040 (E,X) // Biobrick 5

ptet-RBS-RFP-T-T BBa_I13521 (E,S) + linker BBa_B0040 (E,X) // Biobrick 6


nirk (E,S) + RBS-LacI BBa_J24679 (E,X) // Biobrick 7

RBS BBa_J61127 (S,P) + nsr (X,P) // Biobrick 8

ptet BBa_R0040 (S,P) + Biobrick 8 (X,P) // Biobrick 9

placI BBa_K091112 (S,P) + Biobrick 8 (X,P) // Biobrick 10


2nd round

Biobrick 4 (X,P) + RBS-GFP-T-T BBa_I13504 (S,P) // Biobrick 11

Biobrick 7 (X,P) + RBS-RFP-T-T BBa_J04450 (S,P) // Biobrick 12

3rd round

Biobrick 11 + Biobrick 5 // Biobrick 13

Biobrick 11 + Biobrick 6 // Biobrick 14

Biobrick 9 (X,P) + RBS-GFP-T-T BBa_I13504 (S,P) // Biobrick 15

Biobrick 10 (X,P) + RBS-GFP-T-T BBa_I13504 (S,P) // Biobrick 16

4th round

Biobrick 12 + Biobrick 15 // Biobrick 17

Biobrick 12 + Biobrick 16 // Biobrick 18


Biobricks used

BBa_R0040 http://partsregistry.org/wiki/index.php?title=Part:BBa_R0040 ptet P1 6I

BBa_J04450 http://partsregistry.org/wiki/index.php?title=Part:BBa_J04450 pLacI-RBS-RFP-T-T P1 7A

BBa_J24679 http://partsregistry.org/wiki/index.php?title=Part:BBa_J24679 RBS-LacI P1 7L

BBa_J61127 http://partsregistry.org/wiki/index.php?title=Part:BBa_J61127 RBS P1 11N

BBa_E0840 http://partsregistry.org/wiki/index.php?title=Part:BBa_E0840 RBS-GFP-T-T P1 12O

BBa_P0140 http://partsregistry.org/wiki/index.php?title=Part:BBa_P0140 RBS-tetR P2 1F

BBa_P0340 http://partsregistry.org/wiki/index.php?title=Part:BBa_P0340 RBS-tetR P2 1J

BBa_K091112 http://partsregistry.org/wiki/index.php?title=Part:BBa_K091112 placI P2 8B

BBa_K093005 http://partsregistry.org/wiki/index.php?title=Part:BBa_K093005 RBS-RFP-T-T P3 13K

Biobrick digestion

D1.jpg

Agarose gel digestion purification.

From left side to right: BBa_J04450 digested and no digested; BBa_I13521 digested y no digested; lambda-DNA hinIII; nirk ; nsr; phoa

D2.jpg

Agarose gel digestion purification.

From left side to right: BBa_B0014 digestedand no digested; lambda-DNA hinIII; BBa_J61127 digested and no digested; lambda-DNA hinIII ; nirk ; BBa_B0040 digested and no digested.

D3.jpg


Agarose gel digestion purification.

From left side to right: BBa_E0840 digested and no digested; lambda-DNA hinIII; BBa_I13504 digested and no digested; lambda-DNA hinIII; nirk ; BBa_K093005 digested and no digested.

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