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Team:TUDelft/Conjugation Results
From 2009.igem.org
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):
| Data Set | Conjugation Efficiency |
| 1 | 0.028 |
| 2 | 0.028 |
| 3 | 0.025 |
Verification and Characterization of trbK entry exclusion protein
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 1: DH5 α cells with R751 and [http://partsregistry.org/Part:BBa_J23100 J23100] (TRI+AMP)
Transconjugants 2: 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):
| 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 [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.
R751 Gene Knockouts
