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.

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 [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

The first two knockout attempts [1] [2] failed for both oriTR and trbK knockouts. A third attempt is currently in progress using the [http://www.genebridges.com/gb/details.php?prod_id=K004&main_group=red Quick & Easy Conditional Knock Out Kit - FRT] from Gene Bridges.

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