Team:TUDelft/Conjugation Results
From 2009.igem.org
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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. | 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. | ||
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+ | {|border = "0" | ||
+ | |- | ||
+ | |rowspan="4"| | ||
+ | <gallery widths="235px" perrow="2" heights="188px" > | ||
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+ | Image:Conjugation-aug18-trbK_D3tr.jpeg|Set 1 Donors 10<sup>-2</sup> dilution plate | ||
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+ | Image:Conjugation-aug18-trbK_T1tr.jpeg|Set 1 Transconjugants 10<sup>0</sup> dilution plate | ||
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+ | Image:Conjugation-aug18-trbK_D8tr.jpeg|Set 2 Donors 10<sup>-2</sup> dilution plate | ||
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+ | Image:Conjugation-aug18-trbK_T6tr.jpeg|Set 2 Transconjugants 10<sup>0</sup> dilution plate | ||
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+ | </gallery> | ||
+ | |} | ||
==Verification and Characterization of Conjugation Testing Plasmid 1== | ==Verification and Characterization of Conjugation Testing Plasmid 1== |
Latest revision as of 23:03, 21 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 |
|
Verification and Characterization of trbK entry exclusion protein
A conjugation test was done using our conjugation protocol to verify that expression of trbK blocked conjugation.
Donors: NCCBNr# 2350 cells with R751 (TRI)
Receivers: DH5 α cells expressing trbK (CAM)
Transconjugants: DH5 α cells with R751 and expressing trbK (TRI+CAM)
Receivers Control: DH5 α cells with I13522 (AMP)
Transconjugants Control: DH5 α cells with R751 and 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):
Set # | Conjugation Efficinecy |
trbK 1 | 4.49E-6 |
trbK 2 | 2.89E-6 |
control | 0.031 |
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 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 A: DH5 α cells with R751 and J23100 (TRI+AMP)
Transconjugants B: 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 afterward. But if a receiver cell gets R751, it will not accept Conjugation Testing Plasmid 1 afterward because trbK expression will block incoming transfers.
This test will be redone with R751 ΔoriTR cells once they are available.
|
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 Quick & Easy Conditional Knock Out Kit - FRT from Gene Bridges.