Talk:Synthetic Biology:Vectors/Parts/Synthesis order

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Synthesis order check

(by Austin on version ReshmaBioBricksVector20060720.zip)

  • I50040 (pSC101 origin)
    • BioBricks ends
    • Blasted against pSB4A3
    • I50040 contains more of the pSC101 than is present in pSB4A3 (not minimal?)
    • Extra unannotated 11bp sequence at end (not sure it's function)
      • Reshma 15:50, 9 August 2006 (EDT): This is a BaeI site that can be used to cut up the vector if necessary.
    • all mutations in repA coding sequence are silent
    • other mutations appear reasonable
    • Still contains a Nt.BstNBI site in par region. As using this for topoisomerase, seems worth risk to eliminate it
      • Reshma 00:41, 27 July 2006 (EDT): But this is a nicking enzyme and therefore as long as there is not another recognition site nearby, it should be no problem right?
      • Austin 11:41, 27 July 2006 (EDT): It may not be a problem but why leave it so 'messy'? I think it's the only remaining N.BstNBI site. Actually I take that back. There's one in the pUC origin. It seems to me if you're designing the plasmid to use those sites, you may as well clean out all these sites. It's different from cleaning a random nicking site which you don't know whether you'll use in the future. It also may make some things messier. Who knows what would happen if you tried gel purifying after nicking. The ~500bp piece may or may not run with its complementary strand.
      • Reshma 15:22, 9 August 2006 (EDT): Removed. (Against my better judgement.)
  • I51000 (scaffold)
    • BioBricks ends
    • topo + nick site on both sides: ok
    • VF2, VR: ok
    • 2 NheI sites around ampR for cloning: ok
    • ampR mutations do not change coding sequence (compared with pSB1A*)
    • RBS/promoter for ampR not annotated (present in pSB4A)
    • Unclear why VF+ampR-F primer binding site are present (especially as VF would be cut out with NheI)
      • Reshma 00:53, 27 July 2006 (EDT): I think this was a byproduct of my not being entirely sure where the promoter is for ampicillin resistance. I wasn't sure if the VF and ampR-F primer binding site might actually include a part of the promoter driving transcription of ampicillin resistance.
        • Austin 11:41, 27 July 2006 (EDT): As I mentioned above and below, the promoter is annotated in pSB4A and you're including over 30 bp beyond the -35. It seems highly unlikely that it's necessary.
          • Reshma 15:08, 9 August 2006 (EDT): I talked to Tom and the VF sequence was a sequence that was already present in the vector that he chose to be a primer binding site. In fact, the -35 site of AmpR overlaps with the VF sequence. So I've left this part unchanged given that bases upstream of the -35 can affect promoter strength. The exact start points from all promoters in the Registry are somewhat arbitrary.
    • promoter ccdB looks good, ccdB amino acid sequence unchanged
    • pUC origin matches origin from pSB1 except for annotated mutations. not CDS, cannot be certain about effect of mutations
  • P1000 (CmR)
    • BB ends
    • CmR AA seq: ok
    • End includes extra sequence after CmR (would recommend removing it, serves no purpose, is side product of primers TK had to amplify it)
      • Reshma 00:53, 27 July 2006 (EDT): Agreed.
      • Reshma 13:06, 9 August 2006 (EDT): Done.
  • P1001 (TetR)
    • BB ends
    • One base T deletion in pre-promoter region relative to pSB1AT. Already know that P1001 works, so not big deal.
      • Reshma 00:53, 27 July 2006 (EDT): Did you make P1001 and test it out? I don't remember.
      • Austin 11:41, 27 July 2006 (EDT): Yes I have made P1001 and it works.
      • Reshma 13:10, 9 August 2006 (EDT): Reinserted the deleted T for consistency.
    • TetR AA seq: ok
  • P1002 (AmpR)
    • BB ends
    • matches sequence exactly as in scaffold
    • same as above that promoter sequence seems like it could be shortened (you're including 31 bp more upstream from -35)
      • Reshma 00:53, 27 July 2006 (EDT): See above note.
      • Reshma 13:06, 9 August 2006 (EDT): Deleted an extra C at the end of the sequence.
  • P1003 (KanR)
    • BB ends
    • Can definitely remove at least the first 4 bases (they are part of the XhoI site in pSB1AK that was added when cloning it)
      • Reshma 00:53, 27 July 2006 (EDT): Agreed. Did not realize.
      • Reshma 13:06, 9 August 2006 (EDT): Done.
    • KanR AA seq: ok
    • Again End sequence includes extra stuff after stop codon (can't possibly serve any purpose, remove)
      • Reshma 00:53, 27 July 2006 (EDT): Agreed.
      • Reshma 13:06, 9 August 2006 (EDT): Done.
  • pSB4C5-I52000, pSB4K5-I52000, pSB4T5-I52000
    • Didn't recheck sequence, just that they contained the right antibiotic resistance and origin
      • Reshma 00:53, 27 July 2006 (EDT): Not necessary. These molecules are generated from the scaffold and antibiotic resistance markers so they should be identical.

To do

  • Check whether pSC101 and pUC19 are compatible. --> Done!
  1. Cabello F, Timmis K, and Cohen SN. Replication control in a composite plasmid constructed by in vitro linkage of two distinct replicons. Nature. 1976 Jan 29;259(5541):285-90. DOI:10.1038/259285a0 | PubMed ID:765836 | HubMed [Cabello-Nature-1976]
  • Double check that ccdA can be removed from the ccdB operon.
  • Add back in unique restriction sites to enable swapping out of vector pieces.
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