Team:Hawaii/Conjugation Test: Comparing oriT with oriTr
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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.
Methods
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.
Verification/Comparison of oriT
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°C.
- Collect spot and make serial dilutions in LB broth.
- Plate dilutions on LB + Agar plates containing Sm50Sp100, 1mM IPTG and X-GAL.
Results
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.