Team:Paris/August 13

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Line 68: Line 68:
|'''Name'''
|'''Name'''
|'''Ligation'''
|'''Ligation'''
-
|'''Biobricks'''
 
|'''Description'''
|'''Description'''
 +
|'''Biobricks'''
|- style="text-align: center;"
|- style="text-align: center;"
|MP147.1
|MP147.1
|L100.1
|L100.1
| rowspan="3"| rbs TetR - ECFP<br>D110 (BV) -  D130 (BI)
| rowspan="3"| rbs TetR - ECFP<br>D110 (BV) -  D130 (BI)
-
| rowspan="3"| [[Image:Part_icon_rbs.png]][[Image:Icon_coding.png]][[Image:Part_icon_rbs.png]][[Image:Part_icon_reporter.png]][[Image:Part_icon_terminator.png]]
+
| rowspan="3"| [[Image:Part_icon_rbs.png]][[Image:Icon_coding.png]][[Image:Part_icon_rbs.png]][[Image:Part_icon_reporter.png]][[Image:Part_icon_terminator.png]][[Image:Part_icon_terminator.png]]
|-  
|-  
|MP147.2
|MP147.2
Line 85: Line 85:
|L101.1
|L101.1
| rowspan="3"| rbs TetR - GFP tripart<br>D110 (BV) -  D131 (BI)  
| rowspan="3"| rbs TetR - GFP tripart<br>D110 (BV) -  D131 (BI)  
-
| rowspan="3"| [[Image:Part_icon_rbs.png]][[Image:Icon_coding.png]][[Image:Part_icon_rbs.png]][[Image:Part_icon_reporter.png]][[Image:Part_icon_terminator.png]]
+
| rowspan="3"| [[Image:Part_icon_rbs.png]][[Image:Icon_coding.png]][[Image:Part_icon_rbs.png]][[Image:Part_icon_reporter.png]][[Image:Part_icon_terminator.png]][[Image:Part_icon_terminator.png]]
|-  
|-  
|MP148.2
|MP148.2
Line 96: Line 96:
|L114.1
|L114.1
| rowspan="3"| AracpBAD - gfp tripart<br>D126 (BV) - D131 (BI)
| rowspan="3"| AracpBAD - gfp tripart<br>D126 (BV) - D131 (BI)
-
| rowspan="3"| [[Image:Part_icon_regulatory.png]][[Image:Part_icon_reporter.png]]
+
| rowspan="3"| [[Image:Part_icon_regulatory.png]][[Image:Part_icon_rbs.png]][[Image:Part_icon_reporter.png]][[Image:Part_icon_terminator.png]][[Image:Part_icon_terminator.png]]
|-  
|-  
|MP149.2
|MP149.2
Line 107: Line 107:
|L120.1
|L120.1
| rowspan="3"| tetR repressible promoter - ECFP<br>D106 (BV) - D130 (BI)
| rowspan="3"| tetR repressible promoter - ECFP<br>D106 (BV) - D130 (BI)
-
| rowspan="3"| [[Image:Part_icon_regulatory.png]][[Image:Part_icon_rbs.png]][[Image:Part_icon_reporter.png]][[Image:Part_icon_terminator.png]]
+
| rowspan="3"| [[Image:Part_icon_regulatory.png]][[Image:Part_icon_rbs.png]][[Image:Part_icon_reporter.png]][[Image:Part_icon_terminator.png]][[Image:Part_icon_terminator.png]]
|-  
|-  
|MP150.2
|MP150.2
Line 118: Line 118:
|L122.1
|L122.1
| RBS-lasI - ECFP <br>D107 (BV) - D130 (BI)
| RBS-lasI - ECFP <br>D107 (BV) - D130 (BI)
-
| [[Image:Part_icon_rbs.png]][[Image:Icon_coding.png]][[Image:Part_icon_rbs.png]][[Image:Part_icon_reporter.png]][[Image:Part_icon_terminator.png]]
+
| [[Image:Part_icon_rbs.png]][[Image:Icon_coding.png]][[Image:Part_icon_rbs.png]][[Image:Part_icon_reporter.png]][[Image:Part_icon_terminator.png]][[Image:Part_icon_terminator.png]]
|-  
|-  
|MP152.1
|MP152.1
|L123.1
|L123.1
-
| rowspan="3"| RBS lasI - ECFP<br>D107 (BV) - D131 (BI)
+
| rowspan="3"| RBS lasI - gfp tripart<br>D107 (BV) - D131 (BI)
-
| rowspan="3"| [[Image:Part_icon_rbs.png]][[Image:Icon_coding.png]][[Image:Part_icon_reporter.png]][[Image:Part_icon_terminator.png]]
+
| rowspan="3"| [[Image:Part_icon_rbs.png]][[Image:Icon_coding.png]][[Image:Part_icon_rbs.png]][[Image:Part_icon_reporter.png]][[Image:Part_icon_terminator.png]][[Image:Part_icon_terminator.png]]
|-  
|-  
|MP151.2
|MP151.2
Line 144: Line 144:
|MP154.1
|MP154.1
|L132.1
|L132.1
-
| rowspan="3"|
+
| rowspan="3"|flhDC <br> D142(FV) - D139(FI)
-
| rowspan="3"|  
+
| rowspan="3"|[[Image:Icon_coding.png]]
|-  
|-  
|MP154.2
|MP154.2
Line 155: Line 155:
|MP155.1
|MP155.1
|L133.1
|L133.1
-
| rowspan="1"|
+
| rowspan="1"|OmpR*<br> D142(FV) - D140(FI)
-
| rowspan="1"|
+
| rowspan="1"|[[Image:Icon_coding.png]]
|-  
|-  
|MP156.1
|MP156.1
|L134.1
|L134.1
-
| rowspan="3"|
+
| rowspan="3"|EnvZ*<br> D142(FV) - D141(FI)
-
| rowspan="3"|
+
| rowspan="3"|[[Image:Icon_coding.png]]
|-  
|-  
|MP156.2
|MP156.2
Line 171: Line 171:
|MP157.1
|MP157.1
|L138.1
|L138.1
-
| rowspan="1"|
+
| rowspan="1"|gfp generator (E0240)<br> D137(FV) - D138(FI)
-
| rowspan="1"|
+
| rowspan="1"|[[Image:Icon_rbs.png]][[Image:Icon_reporter.png]][[Image:Part_icon_terminator.png]][[Image:Part_icon_terminator.png]]
|}
|}
Line 189: Line 189:
|L100.1
|L100.1
| rowspan="3"| rbs TetR - ECFP<br>D110 (BV) -  D130 (BI)
| rowspan="3"| rbs TetR - ECFP<br>D110 (BV) -  D130 (BI)
-
| rowspan="3"| [[Image:Part_icon_rbs.png]][[Image:Icon_coding.png]][[Image:Part_icon_rbs.png]][[Image:Part_icon_reporter.png]][[Image:Part_icon_terminator.png]]
+
| rowspan="3"| [[Image:Part_icon_rbs.png]][[Image:Icon_coding.png]][[Image:Part_icon_rbs.png]][[Image:Part_icon_reporter.png]][[Image:Part_icon_terminator.png]][[Image:Part_icon_terminator.png]]
|-  
|-  
|S146.2
|S146.2
Line 200: Line 200:
|L101.1
|L101.1
| rowspan="3"| rbs TetR - GFP tripart<br>D110 (BV) -  D131 (BI)  
| rowspan="3"| rbs TetR - GFP tripart<br>D110 (BV) -  D131 (BI)  
-
| rowspan="3"| [[Image:Part_icon_rbs.png]][[Image:Icon_coding.png]][[Image:Part_icon_rbs.png]][[Image:Part_icon_reporter.png]][[Image:Part_icon_terminator.png]]
+
| rowspan="3"| [[Image:Part_icon_rbs.png]][[Image:Icon_coding.png]][[Image:Part_icon_rbs.png]][[Image:Part_icon_reporter.png]][[Image:Part_icon_terminator.png]][[Image:Part_icon_terminator.png]]
|-  
|-  
|S147.2
|S147.2
Line 222: Line 222:
|L120.1
|L120.1
| rowspan="3"| tetR repressible promoter - ECFP<br>D106 (BV) - D130 (BI)
| rowspan="3"| tetR repressible promoter - ECFP<br>D106 (BV) - D130 (BI)
-
| rowspan="3"| [[Image:Part_icon_regulatory.png]][[Image:Part_icon_rbs.png]][[Image:Part_icon_reporter.png]][[Image:Part_icon_terminator.png]]
+
| rowspan="3"| [[Image:Part_icon_regulatory.png]][[Image:Part_icon_rbs.png]][[Image:Part_icon_reporter.png]][[Image:Part_icon_terminator.png]][[Image:Part_icon_terminator.png]]
|-  
|-  
|S149.2
|S149.2
Line 233: Line 233:
|L122.1
|L122.1
| RBS-lasI - ECFP <br>D107 (BV) - D130 (BI)
| RBS-lasI - ECFP <br>D107 (BV) - D130 (BI)
-
| [[Image:Part_icon_rbs.png]][[Image:Icon_coding.png]][[Image:Part_icon_rbs.png]][[Image:Part_icon_reporter.png]][[Image:Part_icon_terminator.png]]
+
| [[Image:Part_icon_rbs.png]][[Image:Icon_coding.png]][[Image:Part_icon_rbs.png]][[Image:Part_icon_reporter.png]][[Image:Part_icon_terminator.png]][[Image:Part_icon_terminator.png]]
|-  
|-  
|S151.1
|S151.1
|L123.1
|L123.1
| rowspan="3"| RBS lasI - ECFP<br>D107 (BV) - D131 (BI)
| rowspan="3"| RBS lasI - ECFP<br>D107 (BV) - D131 (BI)
-
| rowspan="3"| [[Image:Part_icon_rbs.png]][[Image:Icon_coding.png]][[Image:Part_icon_reporter.png]][[Image:Part_icon_terminator.png]]
+
| rowspan="3"| [[Image:Part_icon_rbs.png]][[Image:Icon_coding.png]][[Image:Part_icon_reporter.png]][[Image:Part_icon_terminator.png]][[Image:Part_icon_terminator.png]]
|-  
|-  
-
|S152.2
+
|S151.2
|L123.2
|L123.2
|-  
|-  
-
|S152.3
+
|S151.3
|L123.3
|L123.3
|-  
|-  
Line 250: Line 250:
| rowspan="3"| Strongest RBS (1)- LasR activator (+LVA)<br>D102 (BV) - D114 (BI)
| rowspan="3"| Strongest RBS (1)- LasR activator (+LVA)<br>D102 (BV) - D114 (BI)
| rowspan="3"|[[Image:Part_icon_rbs.png]][[Image:Icon_coding.png]]
| rowspan="3"|[[Image:Part_icon_rbs.png]][[Image:Icon_coding.png]]
 +
|-
 +
|S152.2
 +
|L126.2
 +
|-
 +
|S152.3
 +
|L126.3
|}
|}
-
==PCR screening==
+
==Assay to purify a PCR product using the Qiaquick Gel Extraction kit==
 +
 
 +
*sample used: yesterday PCR product of L130.8 (~40 µL)
 +
*protocol used: Qiagen PCR purification kit
 +
*use of buffer QG (Gel Extraction kit) instead of buffer PBI (PCR purification kit)
 +
*elution in 30 µL of buffer EB
 +
*30 µL of purified product + 12 µL of loading blue
 +
*electrophoresis, 10 µL loaded
 +
=> see '''Gel 1''', well n° 8
 +
 
 +
==> results: PCR products can be purified using the Qiaquick Gel Extraction kit. We just have to replace the buffer PBI by the buffer QG!
 +
 
 +
== '''PCR screening'''==
 +
 
 +
=== Electrophoresis Setting ===
4 more transformants of L130 (pFlhB into J61002) are screened by PCR.
4 more transformants of L130 (pFlhB into J61002) are screened by PCR.
-
*PCR screening programm
+
*PCR screening programm; elogation time: 1 min 30
-
*elogation time : 1 min 30
+
*template: colonies from the transformation plate
*template: colonies from the transformation plate
*positive control: S142 (J61002)
*positive control: S142 (J61002)
Line 262: Line 281:
 +
===Results of Electrophoresis===
 +
 +
[[Image:Screening-PCR-080813.JPG|thumb|'''Gel 1''': PCR screening ('''2-7''') & PCR product purification using the Qiaquick Gel Extraction kit ('''8''')]]
 +
 +
*1% agarose gel
 +
*10 µL loaded
 +
 +
{| style="text-align: center;" border="1"
 +
|'''well n°'''
 +
|1
 +
|2
 +
|3
 +
|4
 +
|5
 +
|6
 +
|7
 +
|8
 +
|-
 +
|'''sample'''
 +
|100 bp DNA ladder
 +
|positive control
 +
|negative control
 +
|'''L130.3'''
 +
|'''L130.4'''
 +
|'''L130.5'''
 +
|'''L130.6'''
 +
|PCR product (L130.8) purified by the Qiaquick Gel Extraction kit
 +
|-
 +
|'''red fluorescence'''
 +
|rowspan="3"|
 +
|strain a little bit pink
 +
|not concerned
 +
|no
 +
|no
 +
|no
 +
|no
 +
|rowspan="3"|not concerned
 +
|-
 +
|'''expected size'''
 +
|1,161 kb
 +
|0 kb
 +
|colspan="4"|<center>'''1,338 kb'''</center>
 +
|-
 +
|'''measured size'''
 +
|style="background: #cbff7B"| 1,2 kb
 +
|style="background: #cbff7B"| 0 kb
 +
|style="background: #ff6d73"| 1,2 kb
 +
|style="background: #ff6d73"| 1,2 kb
 +
|style="background: #ff6d73"| 1,2 kb
 +
|style="background: #ff6d73"| 1,2 kb
 +
|}
 +
==> The L130 transformants analysed are not correct.
-
==Promoter Characterization: work plan==
+
=='''Transformation of the ligation we did [[Team:Paris/August_12|yesterday]]'''==
 +
We transformed L 139, L140, L141 and L142 following the [[Team:Paris/Notebook/Protocols#Transformation|standard protocol using Invitrogen's TOP 10 chemically competent cells]]. The positive control is a transformation with pUC19 and the negative control has no plasmid.

Latest revision as of 16:12, 15 August 2008

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Contents

Sequencing Minipreps we did yesterday

  • O18 has been diluted to have a concentration of 5µM
  • In each tube : 1µL of O18 diluted
  • Pure water qsp 15µL


Name Ligation Concentration MP (µg/mL) Vol MP (µL) Vol H2O (µL) n° tube
MP144.3 L128.3 163 3.68 10.32 AD1
MP144.4 L128.4 154 3.90 10.10 AD2
MP145.6 L129.6 163 3.68 10.32 AD3
MP145.7 L129.7 188 3.19 10.81 AD4
MP146.1 L130.1 298 2.01 11.99 AD5
MP146.2 L130.2 142 4.23 9.77 AD6

Minipreps: Plasmid extraction

  • Protocol (see # 3) Experiments done by QIAcube
  • List of Minipreps
Name Ligation Description Biobricks
MP147.1 L100.1 rbs TetR - ECFP
D110 (BV) - D130 (BI) 		Part icon rbs.pngIcon coding.pngPart icon rbs.pngPart icon reporter.pngPart icon terminator.pngPart icon terminator.png
MP147.2 L100.2
MP147.3 L100.3
MP148.1 L101.1 rbs TetR - GFP tripart
D110 (BV) - D131 (BI) 		Part icon rbs.pngIcon coding.pngPart icon rbs.pngPart icon reporter.pngPart icon terminator.pngPart icon terminator.png
MP148.2 L101.2
MP148.3 L101.3
MP149.1 L114.1 AracpBAD - gfp tripart
D126 (BV) - D131 (BI) 		Part icon regulatory.pngPart icon rbs.pngPart icon reporter.pngPart icon terminator.pngPart icon terminator.png
MP149.2 L114.2
MP149.3 L114.3
MP150.1 L120.1 tetR repressible promoter - ECFP
D106 (BV) - D130 (BI) 		Part icon regulatory.pngPart icon rbs.pngPart icon reporter.pngPart icon terminator.pngPart icon terminator.png
MP150.2 L120.2
MP150.3 L120.3
MP151.1 L122.1 RBS-lasI - ECFP
D107 (BV) - D130 (BI) 		Part icon rbs.pngIcon coding.pngPart icon rbs.pngPart icon reporter.pngPart icon terminator.pngPart icon terminator.png
MP152.1 L123.1 RBS lasI - gfp tripart
D107 (BV) - D131 (BI) 		Part icon rbs.pngIcon coding.pngPart icon rbs.pngPart icon reporter.pngPart icon terminator.pngPart icon terminator.png
MP151.2 L123.2
MP152.3 L123.3
MP153.1 L126.1 Strongest RBS (1)- LasR activator (+LVA)
D102 (BV) - D114 (BI) 		Part icon rbs.pngIcon coding.png
MP153.2 L126.2
MP153.3 L126.3
MP154.1 L132.1 flhDC
D142(FV) - D139(FI) 		Icon coding.png
MP154.2 L132.2
MP154.3 L132.3
MP155.1 L133.1 OmpR*
D142(FV) - D140(FI) 		Icon coding.png
MP156.1 L134.1 EnvZ*
D142(FV) - D141(FI) 		Icon coding.png
MP156.2 L134.2
MP156.3 L134.3
MP157.1 L138.1 gfp generator (E0240)
D137(FV) - D138(FI) 		Icon rbs.pngIcon reporter.pngPart icon terminator.pngPart icon terminator.png

Glycerol Stocks

  • List of Stocks
Strain Ligation Biobricks Description
S146.1 L100.1 rbs TetR - ECFP
D110 (BV) - D130 (BI) 		Part icon rbs.pngIcon coding.pngPart icon rbs.pngPart icon reporter.pngPart icon terminator.pngPart icon terminator.png
S146.2 L100.2
S146.3 L100.3
S147.1 L101.1 rbs TetR - GFP tripart
D110 (BV) - D131 (BI) 		Part icon rbs.pngIcon coding.pngPart icon rbs.pngPart icon reporter.pngPart icon terminator.pngPart icon terminator.png
S147.2 L101.2
S147.3 L101.3
S148.1 L114.1 AracpBAD - gfp tripart
D126 (BV) - D131 (BI) 		Part icon regulatory.pngPart icon reporter.png
S148.2 L114.2
S148.3 L114.3
S149.1 L120.1 tetR repressible promoter - ECFP
D106 (BV) - D130 (BI) 		Part icon regulatory.pngPart icon rbs.pngPart icon reporter.pngPart icon terminator.pngPart icon terminator.png
S149.2 L120.2
S149.3 L120.3
S150.1 L122.1 RBS-lasI - ECFP
D107 (BV) - D130 (BI) 		Part icon rbs.pngIcon coding.pngPart icon rbs.pngPart icon reporter.pngPart icon terminator.pngPart icon terminator.png
S151.1 L123.1 RBS lasI - ECFP
D107 (BV) - D131 (BI) 		Part icon rbs.pngIcon coding.pngPart icon reporter.pngPart icon terminator.pngPart icon terminator.png
S151.2 L123.2
S151.3 L123.3
S152.1 L126.1 Strongest RBS (1)- LasR activator (+LVA)
D102 (BV) - D114 (BI) 		Part icon rbs.pngIcon coding.png
S152.2 L126.2
S152.3 L126.3

Assay to purify a PCR product using the Qiaquick Gel Extraction kit

  • sample used: yesterday PCR product of L130.8 (~40 µL)
  • protocol used: Qiagen PCR purification kit
  • use of buffer QG (Gel Extraction kit) instead of buffer PBI (PCR purification kit)
  • elution in 30 µL of buffer EB
  • 30 µL of purified product + 12 µL of loading blue
  • electrophoresis, 10 µL loaded

=> see Gel 1, well n° 8

==> results: PCR products can be purified using the Qiaquick Gel Extraction kit. We just have to replace the buffer PBI by the buffer QG!

PCR screening

Electrophoresis Setting

4 more transformants of L130 (pFlhB into J61002) are screened by PCR.

  • PCR screening programm; elogation time: 1 min 30
  • template: colonies from the transformation plate
  • positive control: S142 (J61002)
  • negative control: no template
  • primers: O18 and O19


Results of Electrophoresis

Gel 1: PCR screening (2-7) & PCR product purification using the Qiaquick Gel Extraction kit (8)
  • 1% agarose gel
  • 10 µL loaded
well n° 1 2 3 4 5 6 7 8
sample 100 bp DNA ladder positive control negative control L130.3 L130.4 L130.5 L130.6 PCR product (L130.8) purified by the Qiaquick Gel Extraction kit
red fluorescence strain a little bit pink not concerned no no no no not concerned
expected size 1,161 kb 0 kb
1,338 kb
measured size 1,2 kb 0 kb 1,2 kb 1,2 kb 1,2 kb 1,2 kb

==> The L130 transformants analysed are not correct.

Transformation of the ligation we did yesterday

We transformed L 139, L140, L141 and L142 following the standard protocol using Invitrogen's TOP 10 chemically competent cells. The positive control is a transformation with pUC19 and the negative control has no plasmid.

Retrieved from "http://2008.igem.org/Team:Paris/August_13"