Team:TUDelft/Conjugation Results

From 2009.igem.org

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(→Verification and Characterization of Wild R751)
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'''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>
'''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>
'''Receivers''': DH5 &alpha; cells with [http://partsregistry.org/Part:BBa_J23100 J23100] (AMP) <br>
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'''Transconjugants 1''': DH5 &alpha; cells with R751 and [http://partsregistry.org/Part:BBa_J23100 J23100] (TRI+AMP)<br>
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'''Transconjugants A''': DH5 &alpha; cells with R751 and [http://partsregistry.org/Part:BBa_J23100 J23100] (TRI+AMP)<br>
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'''Transconjugants 2''': 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>
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'''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]):
The following table shows the calculated conjugation efficiencies (raw data [https://2009.igem.org/Team:TUDelft/19_August_2009#Calin here]):
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This test will be redone with R751 &Delta;oriTR cells once they are available.
This test will be redone with R751 &Delta;oriTR cells once they are available.
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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" >
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Image:Conjugation-aug18-oriTR_D3oriGFP.jpeg|Set 1 Donors 10<sup>-2</sup> dilution plate
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Image:Conjugation-aug18-oriTR_Ts1oriGFP.jpeg|Set 1 Transconjugants B 10<sup>0</sup> dilution plate
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Image:Conjugation-aug18-oriTR_tl1.jpeg|Set 1 Transconjugants A 10<sup>0</sup> dilution plate
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Image:Conjugation-aug18-oriTR_D8oriGFP.jpeg|Set 2 Donors 10<sup>-2</sup> dilution plate
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Image:Conjugation-aug18-oriTR_Ts6oriGFP.jpeg|Set 2 Transconjugants B 10<sup>0</sup> dilution plate
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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
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</gallery>
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|}

Revision as of 20:33, 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 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 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.

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

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