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From 2011.igem.org

Danyel

• Overview
• To do list

Overview

The constructs involved in the tRNA project so far:

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• 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

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• 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]

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• Proteins with amber mutations
• T7 amber_TT

     S80 : in pSB1AK3

• GFP amber

     [in progress : transformation]

• GFP_TT

     [in progress : transformation]

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• 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.

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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)