Part:BBa_I742123

Multi-host vector pTG262 converted to BioBrick vector

This plasmid was derived from pTG262 by insertion of a biobrick encoding Red Fluorescent Protein between the unique EcoRI and PstI restriction sites. This eliminated the only native XbaI site present in the plasmid, and since there are no native SpeI sites, the resulting plasmid is a compliant BioBrick vector. New biobricks can be inserted into this vector by replacement of the RFP biobrick, and selection of the white colonies.The parent plasmid pTG262 has a multi host replication origin and replicates in a range of gram positive and gram negative bacteria including E. coli, Bacillus subtilis, and Lactobacillus and Lactococcus spp. We hope that it will also replicate in other Gram negative bacteria. At the time of writing we have successfully transformed E. coli and B. subtilis with this construct, selecting with chloramphenicol (15 and 10 mg/l respectively). However, RFP did not seem to be expressed in B. subtilis; possibly a promoter issue.

More information about pTG262 may be found on 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 electroporation 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
  COMPATIBLE WITH RFC[10]
• 12
  INCOMPATIBLE WITH RFC[12]

  Plasmid lacks a prefix.
  Plasmid lacks a suffix.
  Illegal EcoRI site found at 5543
  Illegal NheI site found at 694
  Illegal SpeI site found at 2
  Illegal PstI site found at 16
  Illegal NotI site found at 9
  Illegal NotI site found at 5549
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Plasmid lacks a prefix.
  Plasmid lacks a suffix.
  Illegal EcoRI site found at 5543
  Illegal BglII site found at 3754
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal prefix found at 5543
  Illegal suffix found at 2
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal prefix found at 5543
  Plasmid lacks a suffix.
  Illegal XbaI site found at 5558
  Illegal SpeI site found at 2
  Illegal PstI site found at 16
  Illegal AgeI site found at 3365
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Plasmid lacks a prefix.
  Plasmid lacks a suffix.
  Illegal SapI.rc site found at 3578