Conjugation Results

Verification and Characterization of Wild R751

A conjugation test was done using our conjugation protocol to determine wild R751 conjugation efficiency.

Donors: [http://www.cbs.knaw.nl/databases/nccb/search_bac_plas.aspx NCCBNr# 2350] cells with R751 (TRI)
Receivers: DH5 α cells with [http://partsregistry.org/Part:BBa_I13522 I13522] (AMP)
Transconjugants: DH5 α cells with R751 and [http://partsregistry.org/Part:BBa_I13522 I13522] (TRI+AMP)

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

Set 1 Donors 10-2 dilution plate Set 1 Transconjugants 10-2 dilution plate Set 2 Donors 10-2 dilution plate Set 2 Transconjugants 10-2 dilution plate Set 3 Donors 10-2 dilution plate Set 3 Transconjugants 10-2 dilution plate

Verification and Characterization of trbK entry exclusion protein

A conjugation test was done using our conjugation protocol to verify that expression of [http://partsregistry.org/wiki/index.php?title=Part:BBa_K175001 trbK] blocked conjugation.

Donors: [http://www.cbs.knaw.nl/databases/nccb/search_bac_plas.aspx NCCBNr# 2350] cells with R751 (TRI)
Receivers: DH5 α cells expressing [http://partsregistry.org/wiki/index.php?title=Part:BBa_K175001 trbK] (CAM)
Transconjugants: DH5 α cells with R751 and expressing [http://partsregistry.org/wiki/index.php?title=Part:BBa_K175001 trbK] (TRI+CAM)
Receivers Control: DH5 α cells with [http://partsregistry.org/Part:BBa_I13522 I13522] (AMP)
Transconjugants Control: DH5 α cells with R751 and [http://partsregistry.org/Part:BBa_I13522 I13522] (TRI+AMP)

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

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 [http://partsregistry.org/wiki/index.php?title=Part:BBa_K175005 Conjugation Testing Plasmid 1] was transmitted during conjugation.

Donors: [http://www.cbs.knaw.nl/databases/nccb/search_bac_plas.aspx NCCBNr# 2350] cells with R751 and [http://partsregistry.org/wiki/index.php?title=Part:BBa_K175005 Conjugation Testing Plasmid 1] (TRI+CAM)
Receivers: DH5 α cells with [http://partsregistry.org/Part:BBa_J23100 J23100] (AMP)
Transconjugants A: DH5 α cells with R751 and [http://partsregistry.org/Part:BBa_J23100 J23100] (TRI+AMP)
Transconjugants B: DH5 α cells with [http://partsregistry.org/wiki/index.php?title=Part:BBa_K175005 Conjugation Testing Plasmid 1] and [http://partsregistry.org/Part:BBa_J23100 J23100] (CAM+AMP)

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

The reason for the lower efficiency of [http://partsregistry.org/wiki/index.php?title=Part:BBa_K175005 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 afterwards. But if a receiver cell gets R751, it will not accept Conjugation Testing Plasmid 1 afterwards because trbK expression will block incoming transfers.

This test will be redone with R751 ΔoriTR cells once they are available.

Set 1 Donors 10-2 dilution plate Set 1 Transconjugants B 100 dilution plate Set 1 Transconjugants A 100 dilution plate Set 2 Donors 10-2 dilution plate Set 2 Transconjugants B 100 dilution plate Set 2 Transconjugants A 10-1 dilution plate Set 1 Donors 10-2 dilution plate Set 3 Transconjugants A 10-1 dilution plate

R751 Gene Knockouts