Team:Paris Liliane Bettencourt/Notebook/2011/09/04/

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==Kevin==
==Kevin==
===Biobrick the YFP:tetR fusion protein ===
===Biobrick the YFP:tetR fusion protein ===
-
Let's start again with the good primer to biobrick the protein.
+
Let's start again with good primers to biobrick the protein.
====PCR====
====PCR====
3 Temperatures of primer annealing: 85 - 58 - 48 °C<br>
3 Temperatures of primer annealing: 85 - 58 - 48 °C<br>
-
Primers : pFX234 FW - pFX234 Reverse CP  
+
Primers : pFX234 FW - pFX234 Reverse CP <br>
 +
And PCR purification after
-
====Following steps====
+
[[File:gelpcryfptetr.jpg|450px|thumb|center|Gel of PCR products -> Column 1 and 4 = tetR:YFP]]
-
PCR Purification, digestion in ES, PCR Purification again, Ligation
+
 
 +
====Digestion====
 +
Digestion in ES for S27 and 2 samples of PCR products. PCR Purification again
 +
[[File:geldigyfptetr.jpg|450px|thumb|center|Gel of Digested ES PCR products -> Column 1 and 2 = tetR:YFP - Digested ES S27 -> Column 4]]
 +
It's ok for tetR:YFP digested but we didn't see anything for digested S27.<br>
 +
Do I have made a mistake when I load the gel or the digestion didn't work ? Waiting for transformation to know
 +
 
 +
====Ligation====
 +
1h at microscopy room and storage on freezer in "Sevrès box".
====To be continued====
====To be continued====
Transformation, sequencing and glycerol.
Transformation, sequencing and glycerol.
 +
 +
==Danyel==
 +
*Overview
 +
*To do list
 +
===Overview===
 +
The constructs involved in the tRNA project so far:
 +
 +
----
 +
 +
*'''General information''' : all values are APPROX.
 +
**tRNA = 100 bp
 +
**T7 amber = 2.7 kb
 +
**GFP amber = 720 bp
 +
**pHyperspank = 101 bp
 +
**Pveg_spoVG = 120 bp
 +
**pT7_RBS_GFP_TT = 823 bp
 +
 +
----
 +
 +
*'''tRNA amber suppressor'''
 +
*tRNA
 +
      S38: in pSB1C3
 +
*tRNA_TT
 +
      S58: in pSB1C3
 +
*Pveg_spoVG_tRNA
 +
      S38 in S24 --- [in progress]
 +
*Pveg_spoVG_tRNA_TT
 +
      S81 :  this contruct in pSB1AK3 (ampR)
 +
      S92 :  this contruct in pHM3 (ampR/tetR) --- '''sequencing required''' and transformation in subtilis attempted by Adrien.
 +
*pHyperspank_tRNA 
 +
      S38 in S82 --- [in progress]
 +
*pHyperspank_tRNA_TT
 +
      S58 in S82 --- [in progress]
 +
 +
----
 +
 +
*'''Proteins with amber mutations'''
 +
* T7 amber_TT
 +
      S80 : in pSB1AK3
 +
* GFP amber
 +
      [in progress : transformation]
 +
* GFP_TT
 +
      [in progress : transformation]
 +
 +
----
 +
 +
*'''Other constructs'''
 +
*pT7_RBS_GFP_TT  :
 +
      S79 :  this construct in ampR plasmid (pSB1AK3?).
 +
      *Functionality has been tested through transformation in BL21 and microscopy. (cf.27/08/11)
 +
 +
=== To Do List ===
 +
'''[!]''' You don't have to finish the list! I'm also writing this for myself.
 +
 +
Don't hesitate to refer yourself to the overview.
 +
----
 +
 +
In order to plan ahead.in order of priority:
 +
 +
'''Sequencing'''
 +
*S92 = S81 in pHM3 --- already verified with PCR colony
 +
      Miniprep S92 (labelled in black) in my box.
 +
 +
'''Check negative controls'''
 +
*I've been having trouble with negative controls for the past two weeks. Hence the repetition of this cloning step since.
 +
      If the negative control of S24 shows growth, digestion strategy for S24 needs to be refined.
 +
      The S24 used for yesturday's ligations were gel extracted (c.f.01/09/11)
 +
      '''OR'''  we may be having a problem with one of the glycerols. Minipreps from both glycerols are in my box (S24[x2] and S24A)
 +
 +
'''Cloning'''
 +
*The plates with the orange tape labelled: "Danny's Transfos": c.f. 03/09/11
 +
    1) In case of failure, redo the ligations the transform.
 +
    2) In case of success, all strains are waiting for a strain number.
 +
        ''Verification'' either by sequencing or by PCR Colony.
 +
          (This can be done after the following steps, but preferably before step 3)
 +
        ''S38 in S24'' has to be digested, then ''inserted into pSB1C3''. The others are already in this plasmid.
 +
    3) All constructs must then be ''inserted into pHM3'' and transformed into subtilis.
 +
 +
*The plates with the orange tape labelled: "For Cyrille..."
 +
    1) In case of most probable failure, redo the transformation with the PCR tubes wrapped in orange tape.
 +
      (Box: Extra space)
 +
    2) In case of success,
 +
        Minipreps and glycerols should be made.
 +
        Minipreps should be sent for sequencing.
 +
*S80 in S24
 +
    1) check negative controls for S24 first. Redigest S24 if need be.
 +
    2) Digest S80 with X & P
 +
      Gel migrate (1% - look for 2.7 kb band)
 +
      Gel extract (with isopropanol)
 +
    3) Ligate, transform, plate.
 +
    4) In case of success, name the strain, miniprep & glycerol, followed by PCR colony (optional: sequencing).
 +
    5) Digest and insert into pSB1C3 and pHM3.
 +
    6) Sequencing.
 +
 +
*Optional: retry ligating S38 and S58 into S14 (Pveg with a different RBS)
 +
 +
==Adrien==
 +
 +
-80°C PY79 are resistant to TetR up to 75 μg.mL <sup>-1</sup> which is better than the 150μg.mL <sup>-1</sup> than the -20°C glycerols of PY79. <br> Conclusion: to do efficient electro-poration or chemical transformation into Bacillus and using the multi-host vector pHM3 we must use PY79 from the -80°C glycerol. <br> Tomorrow, Babak will make four glycerol for the -20°C that are going to replace the existing one out of the 4 falcon I launched today.
 +
<br> <br> Cambridge 2008 amylase test was redone with control on the same plate. To be continued by Babak.

Latest revision as of 15:30, 11 September 2011

Contents

Kevin

Biobrick the YFP:tetR fusion protein

Let's start again with good primers to biobrick the protein.

PCR

3 Temperatures of primer annealing: 85 - 58 - 48 °C
Primers : pFX234 FW - pFX234 Reverse CP
And PCR purification after

Gel of PCR products -> Column 1 and 4 = tetR:YFP

Digestion

Digestion in ES for S27 and 2 samples of PCR products. PCR Purification again

Gel of Digested ES PCR products -> Column 1 and 2 = tetR:YFP - Digested ES S27 -> Column 4

It's ok for tetR:YFP digested but we didn't see anything for digested S27.
Do I have made a mistake when I load the gel or the digestion didn't work ? Waiting for transformation to know

Ligation

1h at microscopy room and storage on freezer in "Sevrès box".

To be continued

Transformation, sequencing and glycerol.

Danyel

  • Overview
  • To do list

Overview

The constructs involved in the tRNA project so far:

  • General information : all values are APPROX.
    • tRNA = 100 bp
    • T7 amber = 2.7 kb
    • GFP amber = 720 bp
    • pHyperspank = 101 bp
    • Pveg_spoVG = 120 bp
    • pT7_RBS_GFP_TT = 823 bp
  • tRNA amber suppressor
  • tRNA 
     S38: in pSB1C3
  • tRNA_TT 
     S58: in pSB1C3
  • Pveg_spoVG_tRNA 
     S38 in S24 --- [in progress]
  • Pveg_spoVG_tRNA_TT 
     S81 :  this contruct in pSB1AK3 (ampR)
     S92 :  this contruct in pHM3 (ampR/tetR) --- sequencing required and transformation in subtilis attempted by Adrien.
  • pHyperspank_tRNA 
     S38 in S82 --- [in progress]
  • pHyperspank_tRNA_TT 
     S58 in S82 --- [in progress]
  • Proteins with amber mutations
  • T7 amber_TT 
     S80 : in pSB1AK3
  • GFP amber 
     [in progress : transformation]
  • GFP_TT 
     [in progress : transformation]
  • Other constructs
  • pT7_RBS_GFP_TT  : 
     S79 :  this construct in ampR plasmid (pSB1AK3?).
     *Functionality has been tested through transformation in BL21 and microscopy. (cf.27/08/11)

To Do List

[!] You don't have to finish the list! I'm also writing this for myself.

Don't hesitate to refer yourself to the overview.

In order to plan ahead.in order of priority:

Sequencing

  • S92 = S81 in pHM3 --- already verified with PCR colony 
     Miniprep S92 (labelled in black) in my box.

Check negative controls

  • I've been having trouble with negative controls for the past two weeks. Hence the repetition of this cloning step since. 
     If the negative control of S24 shows growth, digestion strategy for S24 needs to be refined. 
     The S24 used for yesturday's ligations were gel extracted (c.f.01/09/11) 
     OR  we may be having a problem with one of the glycerols. Minipreps from both glycerols are in my box (S24[x2] and S24A)

Cloning

  • The plates with the orange tape labelled: "Danny's Transfos": c.f. 03/09/11 
   1) In case of failure, redo the ligations the transform.
   2) In case of success, all strains are waiting for a strain number.
        Verification either by sequencing or by PCR Colony. 
          (This can be done after the following steps, but preferably before step 3)
        S38 in S24 has to be digested, then inserted into pSB1C3. The others are already in this plasmid.
   3) All constructs must then be inserted into pHM3 and transformed into subtilis.
  • The plates with the orange tape labelled: "For Cyrille..." 
   1) In case of most probable failure, redo the transformation with the PCR tubes wrapped in orange tape.
     (Box: Extra space)
   2) In case of success,
        Minipreps and glycerols should be made.
        Minipreps should be sent for sequencing.
  • S80 in S24 
   1) check negative controls for S24 first. Redigest S24 if need be.
   2) Digest S80 with X & P
      Gel migrate (1% - look for 2.7 kb band)
      Gel extract (with isopropanol)
   3) Ligate, transform, plate.
   4) In case of success, name the strain, miniprep & glycerol, followed by PCR colony (optional: sequencing).
   5) Digest and insert into pSB1C3 and pHM3.
   6) Sequencing.
  • Optional: retry ligating S38 and S58 into S14 (Pveg with a different RBS)

Adrien

-80°C PY79 are resistant to TetR up to 75 μg.mL -1 which is better than the 150μg.mL -1 than the -20°C glycerols of PY79.
Conclusion: to do efficient electro-poration or chemical transformation into Bacillus and using the multi-host vector pHM3 we must use PY79 from the -80°C glycerol.
Tomorrow, Babak will make four glycerol for the -20°C that are going to replace the existing one out of the 4 falcon I launched today.

Cambridge 2008 amylase test was redone with control on the same plate. To be continued by Babak.

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