Team:Hawaii/Conjugation Test: Comparing oriT with oriTr

Conjugation Test: Comparing oriT with oriTr
A plasmid can be made mobilizable by adding the origin of transfer of a self-transmissible plasmid. The [| oriTr] is the relaxation region for the R plasmid (i.e. RP4) and is available as a part in the registry. We have constructed another version of this relaxation region: [| oriT].

We want to compare the efficiencies of these two origins of transfer. To do this, the origins were added to [| LacZ]. After conjugation, the colonies containing the origin of transfer should be identified with blue/white screening and also by antibiotic selection.

Construction of parts:

 * Extraction of oriTr and J33207 from the BioBrick Registry and synthesis of the oriT derived from RP4.
 * Restriction digest followed by ligation into J33207.

Verification of constructs:

 * PCR of colonies with VF2/VR primers to verify size of insert
 * Sequencing of inserts
 * Blue/White Screen to verify that LacZ works.

Conjugation
For conjugation, the mobilizable plasmids (oriTr/J33207 and oriT/J33207)in an E. coli strain will be placed together with a strain of E.coli containing a self-transmissible plasmid (RP4) and also a donor strain which contains a non-mobilizable plasmid which is resistant to a different antibiotic.

After the conjugation the colonies will be plated on selection for the donor strain and IPTG and X-Gal for the blue/white screen and on Sm50Sp100 for antibiotic selection.


 * Grow E. coli in LB with antibiotic selection
 * (mobilizable plasmid)oriTr/J33207 (amp100) in DB3.1
 * (mobilizable plasmid) oriT/J33207 (amp100) in DB3.1
 * (self-transmissible plasmid) RP4 (Kan50) in DH5-a
 * (recipient strain)pSMC121 (Sm50Sp100) in DB3.1


 * Wash donor and recipient strains with LB to remove the antibiotics.


 * Mix donor and recipient strains (50 ul donor (oriTr/J33207 or oriT/J33207), 50 ul donor (RP4), and 100 ul recipient).


 * Centrifuge briefly and discard supernatant.


 * Re-suspend in 10 ul LB broth.


 * Spot on LB + Agar plate with out selection. Incubate 1 hour at 37&deg;C.


 * Collect spot and make serial dilutions in LB broth.


 * Plate dilutions on LB + Agar plates containing Sm50Sp100, 1mM IPTG and X-GAL.

Discussion

 * The experiment was originally designed to only use Blue/White screening to assess the efficiency of conjugation for both of the plasmids (oriTr/J33207 and oriT/J33207), however to give more certainty to the results, antibiotic selection was also added.

Reference

 * The protocol was adapted from [| openwetware.org/wiki/Conjugation], with additional input from Dr. Callahan.