Team:TUDelft/Conjugation Results

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=Conjugation Results=
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='''Conjugation Results'''=
==Verification and Characterization of Wild R751==
==Verification and Characterization of Wild R751==
 +
A conjugation test was done using our [[Team:TUDelft/ConjugationProtocol | 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)<br>
 +
'''Receivers''': DH5 &alpha; cells with [http://partsregistry.org/Part:BBa_I13522 I13522] (AMP) <br>
 +
'''Transconjugants''': DH5 &alpha; cells with R751 and [http://partsregistry.org/Part:BBa_I13522 I13522] (TRI+AMP)<br>
 +
 +
The following table shows the calculated conjugation efficiencies (raw data [https://2009.igem.org/Team:TUDelft/6_August_2009#Calin here]):
 +
 +
{| border="1" align="center"
 +
|- align="center"
 +
| Data Set || Conjugation Efficiency
 +
|- align="center"
 +
| 1 ||  0.028
 +
|- align="center"
 +
| 2 ||  0.028
 +
|- align="center"
 +
| 3 ||  0.025
 +
|}
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 +
{|border = "0"
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|-
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|rowspan="4"|
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<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-T3-GFP.jpg|Set 1 Transconjugants 10<sup>-2</sup> dilution plate
 +
 +
Image:TUDelft-aug5-2009-conj-test-plate-D7-noGFP.jpg|Set 2 Donors 10<sup>-2</sup> dilution plate
 +
 +
Image:TUDelft-aug5-2009-conj-test-plate-T10-GFP.jpg|Set 2 Transconjugants 10<sup>-2</sup> dilution plate
 +
 +
Image:TUDelft-aug5-2009-conj-test-plate-D12-noGFP.jpg|Set 3 Donors 10<sup>-2</sup> dilution plate
 +
 +
Image:TUDelft-aug5-2009-conj-test-plate-T17-GFP.jpg|Set 3 Transconjugants 10<sup>-2</sup> dilution plate
 +
 +
</gallery>
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|}
==Verification and Characterization of trbK entry exclusion protein==
==Verification and Characterization of trbK entry exclusion protein==
 +
A conjugation test was done using our [[Team:TUDelft/ConjugationProtocol | conjugation protocol]] to verify that expression of [http://partsregistry.org/wiki/index.php?title=Part:BBa_K175001 trbK] blocked conjugation.
 +
'''Donors''': [http://www.cbs.knaw.nl/databases/nccb/search_bac_plas.aspx NCCBNr# 2350] cells with R751 (TRI)<br>
 +
'''Receivers''': DH5 &alpha; cells expressing [http://partsregistry.org/wiki/index.php?title=Part:BBa_K175001 trbK] (CAM) <br>
 +
'''Transconjugants''': DH5 &alpha; cells with R751 and expressing [http://partsregistry.org/wiki/index.php?title=Part:BBa_K175001 trbK] (TRI+CAM)<br>
 +
'''Receivers Control''': DH5 &alpha; cells with [http://partsregistry.org/Part:BBa_I13522 I13522] (AMP) <br>
 +
'''Transconjugants Control''': DH5 &alpha; cells with R751 and [http://partsregistry.org/Part:BBa_I13522 I13522] (TRI+AMP)<br>
 +
 +
The positive control test used the same donors but different receivers, as detailed above.
 +
 +
The following table shows the calculated conjugation efficiencies (raw data [https://2009.igem.org/Team:TUDelft/19_August_2009#Calin here]):
 +
 +
{| border="1" align="center"
 +
|- align="center"
 +
| Set # || Conjugation Efficinecy
 +
|- align="center"
 +
| trbK 1 || 4.49E-6
 +
|- align="center"
 +
| trbK 2 || 2.89E-6
 +
|- align="center"
 +
| control || 0.031
 +
|- align="center"
 +
|}
 +
 +
 +
[[Image:TUD_aug18_trbK_efficinecy.png|center]]
 +
 +
 +
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.
 +
 +
{|border = "0"
 +
|-
 +
|rowspan="4"|
 +
<gallery widths="235px" perrow="2" heights="188px" >
 +
 +
Image:Conjugation-aug18-trbK_D3tr.jpeg|Set 1 Donors 10<sup>-2</sup> dilution plate
 +
 +
Image:Conjugation-aug18-trbK_T1tr.jpeg|Set 1 Transconjugants 10<sup>0</sup> dilution plate
 +
 +
Image:Conjugation-aug18-trbK_D8tr.jpeg|Set 2 Donors 10<sup>-2</sup> dilution plate
 +
 +
Image:Conjugation-aug18-trbK_T6tr.jpeg|Set 2 Transconjugants 10<sup>0</sup> dilution plate
 +
 +
</gallery>
 +
|}
==Verification and Characterization of Conjugation Testing Plasmid 1==
==Verification and Characterization of Conjugation Testing Plasmid 1==
 +
A conjugation test was done using our [[Team:TUDelft/ConjugationProtocol | 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)<br>
 +
'''Receivers''': DH5 &alpha; cells with [http://partsregistry.org/Part:BBa_J23100 J23100] (AMP) <br>
 +
'''Transconjugants A''': DH5 &alpha; cells with R751 and [http://partsregistry.org/Part:BBa_J23100 J23100] (TRI+AMP)<br>
 +
'''Transconjugants B''': DH5 &alpha; 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)<br>
 +
 +
The following table shows the calculated conjugation efficiencies (raw data [https://2009.igem.org/Team:TUDelft/19_August_2009#Calin here]):
 +
 +
{| border="1" align="center"
 +
|- align="center"
 +
| Set # || Conjugation Efficiency for Conjugation Testing Plasmid 1 || Conjugation Efficiency for wild R751
 +
|- align="center"
 +
| 1 || 0.00177 || 0.0141
 +
|- align="center"
 +
| 2 || 0.00224 || 0.0476
 +
|- align="center"
 +
| 3|| n/a || 0.0356
 +
|- align="center"
 +
|}
 +
 +
 +
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 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 &Delta;oriTR cells once they are available.
 +
 +
{|border = "0"
 +
|-
 +
|rowspan="4"|
 +
<gallery widths="235px" perrow="2" heights="188px" >
 +
 +
Image:Conjugation-aug18-oriTR_D3oriGFP.jpeg|Set 1 Donors 10<sup>-2</sup> dilution plate
 +
 +
Image:Conjugation-aug18-oriTR_Ts1oriGFP.jpeg|Set 1 Transconjugants B 10<sup>0</sup> dilution plate
 +
 +
Image:Conjugation-aug18-oriTR_tl1.jpeg|Set 1 Transconjugants A 10<sup>0</sup> dilution plate
 +
 +
Image:Conjugation-aug18-oriTR_D8oriGFP.jpeg|Set 2 Donors 10<sup>-2</sup> dilution plate
 +
 +
Image:Conjugation-aug18-oriTR_Ts6oriGFP.jpeg|Set 2 Transconjugants B 10<sup>0</sup> dilution plate
 +
 +
Image:Conjugation-aug18-oriTR_tl7.jpeg|Set 2 Transconjugants A 10<sup>-1</sup> dilution plate
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Image:Conjugation-aug18-oriTR_D13oriGFP.jpeg|Set 1 Donors 10<sup>-2</sup> dilution plate
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Image:Conjugation-aug18-oriTR_tl12.jpeg|Set 3 Transconjugants A 10<sup>-1</sup> dilution plate
 +
 +
</gallery>
 +
|}
==R751 Gene Knockouts==
==R751 Gene Knockouts==
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The first two knockout attempts [[Team:TUDelft/13_August_2009#Calin | [1]]] [[Team:TUDelft/19_August_2009#Calin | [2]]] failed for both oriTR and trbK knockouts. A third attempt is currently in progress using the [http://www.genebridges.com/gb/details.php?prod_id=K004&main_group=red Quick & Easy Conditional Knock Out Kit - FRT] from Gene Bridges.
 +
 +
[[Team:TUDelft | <<< Back Home]]
{{Template:TUDelftiGEM2009_end}}
{{Template:TUDelftiGEM2009_end}}

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: [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

A conjugation test was done using our conjugation protocol to verify that expression of [http://partsregistry.org/wiki/index.php?title=Part:BBa_K175001 trbK] blocked conjugation.

Donors: [http://www.cbs.knaw.nl/databases/nccb/search_bac_plas.aspx NCCBNr# 2350] cells with R751 (TRI)
Receivers: DH5 α cells expressing [http://partsregistry.org/wiki/index.php?title=Part:BBa_K175001 trbK] (CAM)
Transconjugants: DH5 α cells with R751 and expressing [http://partsregistry.org/wiki/index.php?title=Part:BBa_K175001 trbK] (TRI+CAM)
Receivers Control: DH5 α cells with [http://partsregistry.org/Part:BBa_I13522 I13522] (AMP)
Transconjugants Control: DH5 α cells with R751 and [http://partsregistry.org/Part:BBa_I13522 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


TUD aug18 trbK efficinecy.png


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.

Set 1 Donors 10-2 dilution plate

Set 1 Transconjugants 100 dilution plate

Set 2 Donors 10-2 dilution plate

Set 2 Transconjugants 100 dilution plate

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 A: DH5 α cells with R751 and [http://partsregistry.org/Part:BBa_J23100 J23100] (TRI+AMP)
Transconjugants B: 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 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.

Set 1 Donors 10-2 dilution plate

Set 1 Transconjugants B 100 dilution plate

Set 1 Transconjugants A 100 dilution plate

Set 2 Donors 10-2 dilution plate

Set 2 Transconjugants B 100 dilution plate

Set 2 Transconjugants A 10-1 dilution plate

Set 1 Donors 10-2 dilution plate

Set 3 Transconjugants A 10-1 dilution plate

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 [http://www.genebridges.com/gb/details.php?prod_id=K004&main_group=red Quick & Easy Conditional Knock Out Kit - FRT] from Gene Bridges.

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