Team:TUDelft/Conjugation Results

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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 α cells with I13522 (AMP)
Transconjugants: DH5 α cells with R751 and I13522 (TRI+AMP)

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

Data Set Conjugation Efficiency
1 0.028
2 0.028
3 0.025

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 α cells expressing trbK (CAM)
Transconjugants: DH5 α cells with R751 and expressing trbK (TRI+CAM)
Receivers Control: DH5 α cells with I13522 (AMP)
Transconjugants Control: DH5 α 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):

Set # Conjugation Efficinecy
trbK 1 4.49E-6
trbK 2 2.89E-6
control 0.031


TUD aug18 trbK efficinecy.png


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 α cells with J23100 (AMP)
Transconjugants A: DH5 α cells with R751 and J23100 (TRI+AMP)
Transconjugants B: DH5 α cells with Conjugation Testing Plasmid 1 and J23100 (CAM+AMP)

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

Set # Conjugation Efficiency for Conjugation Testing Plasmid 1 Conjugation Efficiency for wild R751
1 0.00177 0.0141
2 0.00224 0.0476
3 n/a 0.0356


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 ΔoriTR 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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