Team:TUDelft/Conjugation Results

=Conjugation Results=

Verification and Characterization of Wild R751
A conjugation test was done using our conjugation protocol to determine wild R751 conjugation efficiency.

Donors: NCCBNr# 2350 cells with R751 (TRI) Receivers: DH5 &alpha; cells with I13522 (AMP) Transconjugants: DH5 &alpha; cells with R751 and I13522 (TRI+AMP)

The following table shows the calculated conjugation efficiencies (raw data here):

Verification and Characterization of trbK entry exclusion protein
A conjugation test was done using our conjugation protocol to verify that expression of trbK blocked conjugation.

Donors: NCCBNr# 2350 cells with R751 (TRI) Receivers: DH5 &alpha; cells expressing trbK (CAM) Transconjugants: DH5 &alpha; cells with R751 and expressing trbK (TRI+CAM) Receivers Control: DH5 &alpha; cells with I13522 (AMP) Transconjugants Control: DH5 &alpha; cells with R751 and I13522 (TRI+AMP)

The positive control test used the same donors but different receivers, as detailed above.

The following table shows the calculated conjugation efficiencies (raw data here):



We can see from this graph that expression of trbK does indeed reduce the conjugation efficiency by four orders of magnitude. With this we have a method to prevent back propagation of the signal and we now have receiver side control over communication.

Verification and Characterization of Conjugation Testing Plasmid 1
A conjugation test was done using our conjugation protocol to verify that Conjugation Testing Plasmid 1 was transmitted during conjugation.

Donors: NCCBNr# 2350 cells with R751 and Conjugation Testing Plasmid 1 (TRI+CAM) Receivers: DH5 &alpha; cells with J23100 (AMP) Transconjugants A: DH5 &alpha; cells with R751 and J23100 (TRI+AMP) Transconjugants B: DH5 &alpha; cells with Conjugation Testing Plasmid 1 and J23100 (CAM+AMP)

The following table shows the calculated conjugation efficiencies (raw data here):

The reason for the lower efficiency of Conjugation Testing Plasmid 1 compared to R751 is the presence of the entry exclusion protein trbK. If a receiver cell gets Conjugation Testing Plasmid 1, it can still receive R751 afterward. But if a receiver cell gets R751, it will not accept Conjugation Testing Plasmid 1 afterward because trbK expression will block incoming transfers.

This test will be redone with R751 &Delta;oriTR cells once they are available.

R751 Gene Knockouts
The first two knockout attempts [1]  [2] failed for both oriTR and trbK knockouts. A third attempt is currently in progress using the Quick & Easy Conditional Knock Out Kit - FRT from Gene Bridges.

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