Melbourne/Plan:Gas vesicles

From 2007.igem.org

Revision as of 05:23, 13 July 2007

Steps:

1. Usefull links (restriction enzymes)
2. Sequences:
   1. Ncbi genebank AF053765 (pNL26 7371bp) (pNL29 6036bp) (original source) (FASTA seq.)
   2. pBluescriptIIKS+ (stratagene) (sequence) (restriction map) (map) (Manual)
      • (no spaces sequence 2961bp)
      • HindIII cuts at 719 ,EcoRI cuts at 707 ,PstI cuts at 701 ,Xbal cuts at 677 ,SpeI cuts at 683
      • (no spaces sequence HindIII *AGCTT....CTGCA* PSTI 2947bp)
   3. pNL26 Plasmid with insert: (seq 10318bp) (res map) (digests)
      1. pNL26 insert PstI--HindIII:(pNL26 7371bp)
   4. pNL29 Plasmid with insert: (seq 8983bp) (res map) (digests)
      1. pNL29 insert PstI--HindIII:(pNL29 6036bp)

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)