Melbourne/Plan:Gas vesicles

From 2007.igem.org

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##*Xbal cuts at 677
##*Xbal cuts at 677
##*SpeI cuts at 683
##*SpeI cuts at 683
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##      pNL26 Plasmid with insert:[[Melbourne/cannon plasmid pNL26 seq| (seq)]] [[Melbourne/cannon plasmid pNL26 res map| (res map)]] [[Melbourne/cannon plasmid pNL26 HindIII digest|(HindIII digest)]]
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##      pNL26 Plasmid with insert:[[Melbourne/cannon plasmid pNL26 seq| (seq 7376)]] [[Melbourne/cannon plasmid pNL26 res map| (res map)]] [[Melbourne/cannon plasmid pNL26 HindIII digest|(HindIII digest)]]
##      pNL29 Plasmid with insert:[[Melbourne/cannon plasmid pNL29 seq| (seq 6041)]] [[Melbourne/cannon plasmid pNL29 res map| (res map)]] [[Melbourne/cannon plasmid pNL29 HindIII digest| (HindIII digest)]]
##      pNL29 Plasmid with insert:[[Melbourne/cannon plasmid pNL29 seq| (seq 6041)]] [[Melbourne/cannon plasmid pNL29 res map| (res map)]] [[Melbourne/cannon plasmid pNL29 HindIII digest| (HindIII digest)]]

Revision as of 21:59, 12 July 2007

Steps:

  1. Sequences:
    1. Ncbi genebank AF053765 (local copy) [http://www.ncbi.nlm.nih.gov/entrez/viewer.fcgi?db=nuccore&id=3089519| (original source)] (FASTA seq.)
    2. pBluescriptIIKS+ [http://www.stratagene.com/products/displayProduct.aspx?pid=267 (stratagene) ][http://www.stratagene.com/vectors/sequences/pbl2ksp_s.txt (sequence) ] [http://www.stratagene.com/vectors/restriction_sites/pbl2ksp_r.txt (restriction map) ][http://www.stratagene.com/vectors/maps/pdf/pBluescript%20II%20KS+_%20webpg.pdf (map) ] [http://www.stratagene.com/manuals/212205.pdf (Manual)]
    3. pNL26 Plasmid with insert: (seq 7376) (res map) (HindIII digest)
    4. pNL29 Plasmid with insert: (seq 6041) (res map) (HindIII digest)


  1. Recovery of genes: (2 days)
    1. Recover the plasmid from paper provided into solution.(method)
    2. Transform E.Coli strain DH5alpha. Screen with Amp 100ug/ml (as per ref.)(electroporation & heatshock)
    3. pick 3 colonies of each
    4. Overnight Culture x9(6 above and 3 form agar block provided)
    5. Miniprep
    6. Produce glycerol stocks
    7. Confirm presence in recovered sample using digest.(HindIII)
    8. ->Established Supply of Plasmid
  1. Induce translation overnight 37deg with IPTG in 100ml plates (method)(2days)
    1. Confirm transcription RT-PRC,
    2. Confirm translation (buoyant phenotype method).
    3. Confirm translation (Namarski optics (direct interferance contrast microscopy method).
  1. Removal of four biobrick like restriction sites all in GvpL.
    1. EcoRI [GAATTC] in gvpL (2858)
    2. PstI [CTGCAG] in gvpL x 3 (2522,2900,3005)
    3. XbaI [TCTAGA] (not present)
    4. SpeI [ACTAGT] (not present)
  1. Insertion of biobrick required restriction sites by PCR primer modification.(method)
    1. Design of primers
    2. Primer generation
    3. Plasmid extraction from culture
    4. PCR
    5. Restriction EcoR1 & Spe1
    6. Gel separation
    7. Restriction of standard Library death plasmid EcoR1,Spe1.
    8. Ligation.
    9. Transform host with regulated POPS output
    10. Confirm dna, rna , protein (as for A)
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