Team:UAB-Barcelona/CS1

From 2009.igem.org

(Difference between revisions)
(New page: {{Template:UAB-Barcelona2}} ==Cloning strategy== ====1st round==== '''phoa''' (E,S) + backbone BBa_K093005 (E,S)  ''Biobrick 1'' '''nirk''' (E,S) + backbone BBa_K093005 (E,S)  ''B...)
 
(13 intermediate revisions not shown)
Line 1: Line 1:
{{Template:UAB-Barcelona2}}
{{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==
==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====
====1st round====
-
'''phoa''' (E,S) + backbone BBa_K093005 (E,S) ''Biobrick 1''
+
'''phoa''' (E,S) + backbone BBa_K093005 (E,S) // ''Biobrick 1''
-
'''nirk''' (E,S) + backbone BBa_K093005 (E,S) ''Biobrick 2''
+
'''nirk''' (E,S) + backbone BBa_K093005 (E,S) // ''Biobrick 2''
-
'''nsr''' (X,P) + backbone BBa_K093005 (E,S) ''Biobrick 3''
+
'''nsr''' (X,P) + backbone BBa_K093005 (E,S) // ''Biobrick 3''
Line 32: Line 45:
''Biobrick 4'' (X,P) + '''RBS-GFP-T-T''' BBa_I13504 (S,P) // ''Biobrick 11''
''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====
====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''
 +
 +
 +
{|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
 +
 +
 +
'''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.

Retrieved from "http://2009.igem.org/Team:UAB-Barcelona/CS1"