Team:UAB-Barcelona/CS1

From 2009.igem.org

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Latest revision as of 03:36, 22 October 2009

Documento sin título

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

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

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.

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.

@@ Line 1: / Line 1: @@
 {{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 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) <html><a href="https://static.igem.org/mediawiki/2009/1/1e/Protocols.pdf">(PROTOCOL)</a></html>.
 ==Cloning strategy==
+{|class="wikitable" border="0" cellpadding="10" cellspacing="1" style="padding: 0; background-color:black;  border: 1px solid black; text-align:justify; margin:5px -5px 5px -5px"
+!width="30%"; valign="top" style="background:#ECC850; color:black"|
 ====1st round====
@@ Line 32: / Line 45: @@
 ''Biobrick 4'' (X,P) + '''RBS-GFP-T-T''' BBa_I13504 (S,P) // ''Biobrick 11''
-''Biobrick 7'' (X,P) + '''RBS-GFP-T-T''' BBa_E0840 (S,P) // ''Biobrick 12''
+''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 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''
+{|class="wikitable" border="0" cellpadding="10" cellspacing="1" style="padding: 0; background-color:black;  border: 1px solid black; text-align:justify; margin:5px -5px 5px -5px"
+!width="30%"; valign="top" style="background:white; color:black"|
+=====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==
+https://static.igem.org/mediawiki/2009/7/77/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
+https://static.igem.org/mediawiki/2009/5/53/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.
+https://static.igem.org/mediawiki/2009/c/ce/D3.jpg
-''Biobrick 11'' + ''Biobrick 6 '' // Biobrick 14
-''Biobrick 9'' (X,P) + '''RBS-GFP-T-T''' BBa_I13504 (S,P) // Biobrick 15
+'''Agarose gel digestion purification.'''
-''Biobrick 10'' (X,P) + '''RBS-GFP-T-T''' BBa_I13504 (S,P) // Biobrick 16
+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.

Team:UAB-Barcelona/CS1

From 2009.igem.org

Latest revision as of 03:36, 22 October 2009

Contents

Transformation with biobricks

Cloning strategy

1st round

2nd round

3rd round

4th round

Biobricks used

Biobrick digestion