Part:BBa_I742103

pTG262 Plasmid (Gram negative to Gram positive shuttle vector)

This plasmid contains an origin of replication which is capable of replicating in E. coli and several gram positive organisms including Lactobacillus, Lactococcus and Bacillus. The plasmid has chloramphenicol, neomycin & gentamicin resistance genes. It also has EcoRI and PstI sites with an XbaI site situated in the middle so biobricks can be inserted. Insertion of any biobrick between the unique EcoRI and PstI sites removes the only XbaI site, and there are no SpeI sites, so the product is a compliant biobrick. pTG262 was generously provided by Dr. Mike Gasson of the Institute of Food Research, Norwich, UK. Recommended selection with chloramphenicol is as follows: E. coli 15 mg/l; Bacillus subtilis 10 mg/l; Lactococcus lactis 5 mg/l; Lactobacillus spp. 7.5 mg/l.

Gene instability may occur with certain inserts (we have not experienced this so far with the biobricks we have inserted).

For more information about pTG262 visit the [http://2007.igem.org/Edinburgh/Yoghurt/Design#Gene_Expression_In_Lactobacillus Edinburgh Yoghurt Wiki]

Usage and Biology

from MIT iGEM Team 2008: When transforming into E. Coli, be sure to plate as much as possible (spin down and resuspend cells) and give 2-3 days for the cells to grow. Watch out for background growth, make sure to verify that you have the actual plasmid. We have found that electroporating this plasmid into E. Coli works well (we use BL21 cells), as the transformation efficiency is improved with less background, and you can see colonies in ~1 day. We have concluded that it is not likely pTG262 can be transformed into Lactobacillus via electroporation. Look at the user review page for more detailed information.

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal EcoRI site found at 463
  Illegal XbaI site found at 436
  Illegal PstI site found at 424
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal EcoRI site found at 463
  Illegal NheI site found at 5313
  Illegal PstI site found at 424
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal EcoRI site found at 463
  Illegal BglII site found at 2252
  Illegal BamHI site found at 442
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal EcoRI site found at 463
  Illegal XbaI site found at 436
  Illegal PstI site found at 424
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal EcoRI site found at 463
  Illegal XbaI site found at 436
  Illegal PstI site found at 424
  Illegal AgeI site found at 2641
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal SapI site found at 2427