Part:BBa_K175005:Experience

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Characterization of BBa_K175005

A conjugation test was done using the TUDelft iGEM 2009 [http://2009.igem.org/Team:TUDelft/ConjugationProtocol conjugation protocol] to verify that K175005 on a pSB4C5 low-copy backbone was transmitted during conjugation.

Donors: [http://www.cbs.knaw.nl/databases/nccb/search_bac_plas.aspx NCCBNr# 2350] cells with R751 and K175005-pSB4C5 (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 K175005-pSB4C5 and J23100 (CAM+AMP)

The following table shows the calculated conjugation efficiencies (raw data [http://2009.igem.org/Team:TUDelft/19_August_2009#Calin here]):

Set #	Conjugation Efficiency for K175005-pSB4C5	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 K175005-pSB4C5 compared to R751 is the presence of the entry exclusion protein trbK. If a receiver cell gets K175005-pSB4C5, it can still receive R751 afterward. But if a receiver cell gets R751, it will not accept K175005-pSB4C5 afterward because trbK expression will block incoming transfers.

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