Team:UAB-Barcelona/CS1

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Revision as of 02:37, 22 October 2009

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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 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).

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

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 {{Template:UAB-Barcelona2}}
+==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 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).
+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>.
 ==Cloning strategy==