Team:TUDelft/Conjugation Results

From 2009.igem.org

(Difference between revisions)
(→Verification and Characterization of Wild R751)
(→Verification and Characterization of Wild R751)
Line 23: Line 23:
| 3 ||  0.025
| 3 ||  0.025
|}
|}
-
 
{|border = "0"
{|border = "0"
|-
|-
|rowspan="4"|
|rowspan="4"|
-
<gallery widths="240px" perrow="2" heights="188px" >
+
<gallery widths="235px" perrow="2" heights="188px" >
Image:TUDelft-aug5-2009-conj-test-plate-D2-noGFP.jpg|Set 1 Donors 10<sup>-2</sup> dilution plate
Image:TUDelft-aug5-2009-conj-test-plate-D2-noGFP.jpg|Set 1 Donors 10<sup>-2</sup> dilution plate

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: NCCBNr# 2350 cells with R751 (TRI)
Receivers: DH5 α cells with I13522 (AMP)
Transconjugants: DH5 α cells with R751 and 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 Conjugation Testing Plasmid 1 was transmitted during conjugation.

Donors: NCCBNr# 2350 cells with R751 and Conjugation Testing Plasmid 1 (TRI+CAM)
Receivers: DH5 α cells with J23100 (AMP)
Transconjugants 1: DH5 α cells with R751 and J23100 (TRI+AMP)
Transconjugants 2: DH5 α cells with Conjugation Testing Plasmid 1 and 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 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

Retrieved from "http://2009.igem.org/Team:TUDelft/Conjugation_Results"