From 2007.igem.org

Steps:

1. Usefull links (restriction enzymes)(Software)<open wetware primer design> <more primer design>
2. Sequences:
   1. Ncbi genebank AF053765 (pNL26 7371bp) (pNL29 6036bp) [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)]
      • (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) (reverse complement)
      1. pNL26 insert PstI--HindIII:(pNL26 7371bp)
   4. pNL29 Plasmid with insert: (seq 8983bp) (res map) (digests) (reverse complement)
      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. [[http://www.kazusa.or.jp/codon/cgi-bin/showcodon.cgi?species=Escherichia+coli+K12+[gbbct]| DNA code Usage in Ecoli K12]]
   2. EcoRI [GAATTC] in gvpL (2858)is out of frame [GA G][AAT][TC A]-> [E(glutamate),N(asparagine),S(serine)]
      1. Replace with [GA A][AAT][TC A] Glutamate: from [GAG](K12 usage 18%) to [GAA](K12 usage 40%).
   3. PstI [CTGCAG] in gvpL x 3 (2522,2900,3005) each is in frame [CTG][CAG]-> [L(leucine),Q(glutamine)]
      1. Replace with [CTG][CAA] Glutamine: [CAG](K12 usage 29%) to [CAA](K12 usage 15%).
   4. XbaI [TCTAGA] (not present)
   5. 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)