Team:Paris/August 25

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(→transformation of the ligations we did yesterday)
(→PCR screenning: transformation results from August 23th)
 
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|D110
|D110
|Amp
|Amp
 +
|}
 +
 +
 +
='''Construction of pFlgA - GFP Generator'''=
 +
 +
Aim : Construction of ''' "pFlgA-RBS-GFP-dbl ter" (pFlgA-E0240)''' [[Image:Part_icon_regulatory.png]][[Image:Part_icon_rbs.png]][[Image:icon_reporter.png]][[Image:Part_icon_terminator.png]][[Image:Part_icon_terminator.png]]
 +
 +
=='''Results of the transformation we did [[Team:Paris/August 23  |the day before yesterday]]'''==
 +
 +
{|border="1" style="text-align: center"
 +
|'''Ligation name'''
 +
|'''Description'''
 +
|'''Antibio'''
 +
|'''Number Colonies observed'''
 +
|'''Fluorescence'''
 +
|'''Comments'''
 +
|-
 +
|colspan="6" |Ligations
 +
|-
 +
|L164
 +
|D168(FV) - D132(FI)<br>pFlgA - gfp generator
 +
|Amp
 +
| 125
 +
| No
 +
| ok
 +
|-
 +
|colspan="6" |Controls
 +
|-
 +
|TL164
 +
|D168(FV)
 +
|Amp
 +
| 8
 +
| No
 +
| ok
 +
|-
 +
|Positive Control
 +
|pUC19
 +
|Amp
 +
|2000 (efficiency 2.10^8)
 +
|No
 +
|OK
 +
|}
 +
 +
=> Need to screen to know which clones we can use for the  of  '''pFlgA promotor characterization'''.
 +
 +
='''Cloning of EnvZ*'''=
 +
The sequencing of EnvZ* previously cloned, revealed a loss of about 300 bp. EnvZ* contains indeed an EcoRI restriction site within its sequence. So we can't use this enzyme during the cloning.
 +
 +
==Digestion==
 +
 +
{|border="1" style="text-align: center"
 +
|'''digestion number'''
 +
|'''name'''
 +
|'''template'''
 +
|'''Enzymes'''
 +
|-
 +
|D159
 +
|EnvZ*
 +
|PCR129 from August 8th
 +
|XbaI & PstI
 +
|-
 +
|D116
 +
|pSB1A2
 +
|MP108 (C0179 (lasR-pSB1A2))
 +
|XbaI & PstI
 +
|-
 +
|}
 +
 +
'''Reaction mixture'''
 +
*4 µL of PCR129 (or 2 µL of MP108)
 +
*3 µL of 10X buffer n°2
 +
*0,3 µL of 100X BSA
 +
*1 µL of XbaI
 +
*1 µL of PstI
 +
*20,7 µL (or 22,7 µL) of water
 +
Incubation at 37°C during 2H25, and then ~20 min at 65°C
 +
 +
==Electrophoresis==
 +
 +
[[Image:KR000225.jpg|thumb|]]
 +
 +
1% agarose gel
 +
*EnvZ*: 3 µL of digestion products + 1 µL of loading blue + 2 µL of water
 +
*pSB1A2: 30 µL of digestion products + 6 µL of loading blue
 +
 +
{|border="1" style="text-align: center"
 +
|'''well n°'''
 +
|1
 +
|2
 +
|3
 +
|4
 +
|5
 +
|6
 +
|-
 +
|'''sample'''
 +
|1 kb DNA ladder
 +
|D159 (EnvZ*)
 +
|nothing
 +
|D116 (pSB1A2 & lasR)
 +
|nothing
 +
|100 bp DNA ladder
 +
|-
 +
|'''expected size'''
 +
|
 +
|1421 bp
 +
|
 +
|2057 bp & 707 bp
 +
|
 +
|
 +
|-
 +
|'''measured size'''
 +
|
 +
|1,4 kb
 +
|
 +
|2 kb & 0.7 kb
 +
|
 +
|
 +
|-
 +
|}
 +
<br>
 +
'''Purification'''
 +
*EnvZ*: digestion product purified directly by Qiagen kit
 +
*pSB1A2: excision of the band (2 kb) from the gel and purification by the Qiaquick Gel Extraction kit
 +
elution in 30 µL of buffer EB
 +
<br>then kept at - 20°C
 +
 +
 +
 +
='''Screening of the cloning of pFlgA-YFP Tripart (LVA+/-)'''=
 +
 +
==Electrophoresis==
 +
[[Image:KR000232.JPG|thumb|Gel n°1 : Screening of L160]]
 +
[[Image:KR000229.JPG|thumb|Gel n°2 : Screening of L161]]
 +
{|border="1" style="text-align: center"
 +
|
 +
|colspan="16"|Gel n° 1
 +
|-
 +
|'''well n°'''
 +
|1
 +
|2
 +
|3
 +
|4
 +
|5
 +
|6
 +
|7
 +
|8
 +
|9
 +
|10
 +
|11
 +
|12
 +
|13
 +
|14
 +
|15
 +
|16
 +
|-
 +
|'''sample'''
 +
|100pb ladder
 +
|colspan="7"|don't matter
 +
|L160.1
 +
|L160.2
 +
|L160.3
 +
|L160.4
 +
|L160.5
 +
|L160.6
 +
|L160.7
 +
|L160.8
 +
|-
 +
|'''expected size (pb)'''
 +
|
 +
|colspan="7"|
 +
|colspan="8"|1 200
 +
|-
 +
|'''measured size (pb)'''
 +
|
 +
|colspan="7"|
 +
|style="background: #cbff7B"|1 200
 +
|style="background: #cbff7B"|1 200
 +
|style="background: #cbff7B"|1 200
 +
|style="background: #cbff7B"|1 200
 +
|style="background: #cbff7B"|1 200
 +
|style="background: #cbff7B"|1 200
 +
|style="background: #cbff7B"|1 200
 +
|style="background: #cbff7B"|1 200
 +
|}
 +
 +
{|border="1" style="text-align: center"
 +
|
 +
|colspan="16"|Gel n° 2
 +
|-
 +
|'''well n°'''
 +
|1
 +
|2
 +
|3
 +
|4
 +
|5
 +
|6
 +
|7
 +
|8
 +
|9
 +
|10
 +
|11
 +
|12
 +
|13
 +
|14
 +
|15
 +
|16
 +
|-
 +
|'''sample'''
 +
|L161.1
 +
|100 pb ladder
 +
|L161.2
 +
|L161.3
 +
|colspan="12"|don't matter
 +
|-
 +
|'''expected size (pb)'''
 +
|1 167
 +
|
 +
|colspan="2"|1 167
 +
|-
 +
|'''measured size (pb)'''
 +
|style="background: #cbff7B"|1 300
 +
|
 +
|style="background: #cbff7B"|1 300
 +
|style="background: #cbff7B"|1 30
 +
|}
 +
 +
==Minipreps and glycerol stock==
 +
 +
{|border="1" style="text-align: center"
 +
|'''Miniprep'''
 +
|'''Glycerol Stock
 +
|'''Ligation'''
 +
|'''Name'''
 +
|-
 +
|MP1
 +
|S1
 +
|L160
 +
|FlgA-rbs-YFP-dbl ter
 +
|-
 +
|MP1
 +
|S1
 +
|L161
 +
|FlgA-rbs-YFP-LVA+-dbl ter
 +
|}
 +
 +
 +
='''Promoter characterization plasmids'''=
 +
 +
==PCR screenning: transformation results from August 23th==
 +
 +
 +
[[Team:Paris/Notebook/Protocols#Transformation |Protocol]]
 +
 +
[[Image:25-08-08.png|200px]]
 +
[[Image:25-08-08bis.png|200px]]
 +
 +
 +
{|border="1" style="text-align: center"
 +
|'''Ligation name'''
 +
|'''Clone number'''
 +
|'''Product description'''
 +
|'''Primers used'''
 +
|'''Size expected'''
 +
|'''Size observed'''
 +
|-
 +
|no name ligation
 +
|1
 +
|tetR-B0015
 +
|O18-O19
 +
|
 +
|
 +
|-
 +
|Control L166
 +
|1
 +
|Vector autoligation control
 +
|O18-O19
 +
|249
 +
|correct
 +
|-
 +
|L166
 +
|1-5,7-9,11-14
 +
|RBS B0032 - tetR
 +
|O18-O19
 +
|
 +
|around 249
 +
|-
 +
|L166
 +
|6
 +
|RBS B0032 - tetR
 +
|O18-O19
 +
|
 +
|
 +
|-
 +
|L166
 +
|10
 +
|RBS B0032 - tetR
 +
|O18-O19
 +
|
 +
|
 +
|-
 +
|L167
 +
|1
 +
|gfp generator - pTet
 +
|O18-O19
 +
|
 +
|
|}
|}

Latest revision as of 19:27, 9 September 2008

← Yesterday

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Contents

Construction for Synchronization

Transformation of the ligations we did yesterday

Ligation Name Description Antibio
L159 D125 (FV) + D109 (FI) Kan
Control L159 D125 Kan
L162 D107 (BV) + D163 (BI) Amp
Control L162 D107 Amp
L163 D110 (BV) + D163 (BI) Amp
Control L163 D110 Amp


Construction of pFlgA - GFP Generator

Aim : Construction of "pFlgA-RBS-GFP-dbl ter" (pFlgA-E0240) Part icon regulatory.pngPart icon rbs.pngIcon reporter.pngPart icon terminator.pngPart icon terminator.png

Results of the transformation we did the day before yesterday

Ligation name Description Antibio Number Colonies observed Fluorescence Comments
Ligations
L164 D168(FV) - D132(FI)
pFlgA - gfp generator 		Amp 125 No ok
Controls
TL164 D168(FV) Amp 8 No ok
Positive Control pUC19 Amp 2000 (efficiency 2.10^8) No OK 
=> Need to screen to know which clones we can use for the  of  pFlgA promotor characterization.

Cloning of EnvZ*

The sequencing of EnvZ* previously cloned, revealed a loss of about 300 bp. EnvZ* contains indeed an EcoRI restriction site within its sequence. So we can't use this enzyme during the cloning.

Digestion

digestion number name template Enzymes
D159 EnvZ* PCR129 from August 8th XbaI & PstI
D116 pSB1A2 MP108 (C0179 (lasR-pSB1A2)) XbaI & PstI

Reaction mixture

  • 4 µL of PCR129 (or 2 µL of MP108)
  • 3 µL of 10X buffer n°2
  • 0,3 µL of 100X BSA
  • 1 µL of XbaI
  • 1 µL of PstI
  • 20,7 µL (or 22,7 µL) of water

Incubation at 37°C during 2H25, and then ~20 min at 65°C

Electrophoresis

KR000225.jpg

1% agarose gel

  • EnvZ*: 3 µL of digestion products + 1 µL of loading blue + 2 µL of water
  • pSB1A2: 30 µL of digestion products + 6 µL of loading blue
well n° 1 2 3 4 5 6
sample 1 kb DNA ladder D159 (EnvZ*) nothing D116 (pSB1A2 & lasR) nothing 100 bp DNA ladder
expected size 1421 bp 2057 bp & 707 bp
measured size 1,4 kb 2 kb & 0.7 kb


Purification

  • EnvZ*: digestion product purified directly by Qiagen kit
  • pSB1A2: excision of the band (2 kb) from the gel and purification by the Qiaquick Gel Extraction kit

elution in 30 µL of buffer EB
then kept at - 20°C


Screening of the cloning of pFlgA-YFP Tripart (LVA+/-)

Electrophoresis

Gel n°1 : Screening of L160
Gel n°2 : Screening of L161
Gel n° 1
well n° 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16
sample 100pb ladder don't matter L160.1 L160.2 L160.3 L160.4 L160.5 L160.6 L160.7 L160.8
expected size (pb) 1 200
measured size (pb) 1 200 1 200 1 200 1 200 1 200 1 200 1 200 1 200
Gel n° 2
well n° 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16
sample L161.1 100 pb ladder L161.2 L161.3 don't matter
expected size (pb) 1 167 1 167
measured size (pb) 1 300 1 300 1 30

Minipreps and glycerol stock

Miniprep Glycerol Stock Ligation Name
MP1 S1 L160 FlgA-rbs-YFP-dbl ter
MP1 S1 L161 FlgA-rbs-YFP-LVA+-dbl ter


Promoter characterization plasmids

PCR screenning: transformation results from August 23th

Protocol

25-08-08.png 25-08-08bis.png


Ligation name Clone number Product description Primers used Size expected Size observed
no name ligation 1 tetR-B0015 O18-O19
Control L166 1 Vector autoligation control O18-O19 249 correct
L166 1-5,7-9,11-14 RBS B0032 - tetR O18-O19 around 249
L166 6 RBS B0032 - tetR O18-O19
L166 10 RBS B0032 - tetR O18-O19
L167 1 gfp generator - pTet O18-O19
Retrieved from "http://2008.igem.org/Team:Paris/August_25"