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 origionally 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.

[http://manoa.hawaii.edu/ ][http://manoa.hawaii.edu/ovcrge/ ][http://www.ctahr.hawaii.edu ]