Melbourne/Plan:Gas vesicles
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Revision as of 01:15, 17 July 2007 by PhillipDodson (Talk | contribs)
Steps:
- Usefull links (restriction enzymes)(Software)[<open wetware primer design>] [<more primer design>]
- Sequences:
- Ncbi genebank AF053765 (pNL26 7371bp) (pNL29 6036bp) (original source) (FASTA seq.)
- 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)
- pNL26 Plasmid with insert: (seq 10318bp) (res map) (digests) (reverse complement)
- pNL26 insert PstI--HindIII:(pNL26 7371bp)
- pNL29 Plasmid with insert: (seq 8983bp) (res map) (digests) (reverse complement)
- pNL29 insert PstI--HindIII:(pNL29 6036bp)
- Recovery of genes: (2 days)
- Recover the plasmid from paper provided into solution.(method)
- Transform E.Coli strain DH5alpha. Screen with Amp 100ug/ml (as per ref.)(electroporation & heatshock)
- pick 3 colonies of each
- Overnight Culture x9(6 above and 3 form agar block provided)
- Miniprep
- Produce glycerol stocks
- Confirm presence in recovered sample using digest.(HindIII)
- ->Established Supply of Plasmid
- Induce translation overnight 37deg with IPTG in 100ml plates (method)(2days)
- Confirm transcription RT-PRC,
- Confirm translation (buoyant phenotype method).
- Confirm translation (Namarski optics (direct interferance contrast microscopy method).
- Removal of four biobrick like restriction sites all in GvpL.
- DNA code Usage in Ecoli K12 from http://www.kazusa.or.jp/codon (result)
- QuickchangeII XL 200522(x30) 200521(x10): Quickchange XL Kit
- EcoRI [GAATTC] in gvpL (2858)is out of frame [GA G][AAT][TC A]-> [E(glutamate),N(asparagine),S(serine)]
- Replace with [GA A][AAT][TC A] Glutamate: from [GAG](K12 usage 18%) to [GAA](K12 usage 40%).
- PstI [CTGCAG] in gvpL x 3 (2522,2900,3005) each is in frame [CTG][CAG]-> [L(leucine),Q(glutamine)]
- Replace with [CTG][CAA] Glutamine: [CAG](K12 usage 29%) to [CAA](K12 usage 15%).
- XbaI [TCTAGA] (not present)
- SpeI [ACTAGT] (not present)
- EcoRI [GAATTC] in gvpL (2858)is out of frame [GA G][AAT][TC A]-> [E(glutamate),N(asparagine),S(serine)]
- Insertion of biobrick required restriction sites by PCR primer modification.(method)
- Design of primers
- Primer generation
- Plasmid extraction from culture
- PCR
- Restriction EcoR1 & Spe1
- Gel separation
- Restriction of standard Library death plasmid EcoR1,Spe1.
- Ligation.
- Transform host with regulated POPS output
- Confirm dna, rna , protein (as for A)