Team:TUDelft/Conjugation Results

From 2009.igem.org

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Image:TUDelft-aug5-2009-conj-test-plate-D2-noGFP.jpg|Set 1 Donors 10<sup>-2</sup> dilution plate
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Image:TUDelft-aug5-2009-conj-test-plate-T3-GFP.jpg|Set 1 Transconjugants 10<sup>-2</sup> dilution plate
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Image:TUDelft-aug5-2009-conj-test-plate-D7-noGFP.jpg|Set 2 Donors 10<sup>-2</sup> dilution plate
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Image:TUDelft-aug5-2009-conj-test-plate-T10-GFP.jpg|Set 2 Transconjugants 10<sup>-2</sup> dilution plate
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Image:TUDelft-aug5-2009-conj-test-plate-D12-noGFP.jpg|Set 3 Donors 10<sup>-2</sup> dilution plate
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Image:TUDelft-aug5-2009-conj-test-plate-T17-GFP.jpg|Set 3 Transconjugants 10<sup>-2</sup> dilution plate
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==Verification and Characterization of trbK entry exclusion protein==
==Verification and Characterization of trbK entry exclusion protein==

Revision as of 20:24, 19 October 2009

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


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

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

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