Melbourne/Plan:Gas vesicles

From 2007.igem.org


Steps:

  1. Recovery of genes:
    1. Recover the plasmid from sample provided into solution.
    2. Transform E.Coli strain DH5alpha. Screen with Amp
    3. Culture->Establish Supply of Plasmid
    4. Confirm presence in recovered sample using digest.(HindIII)
    5. Induce translation IPTG and confirm transcription RT-PRC, and Translation (buoyant phenotype).
  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.
    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)