Team:TUDelft/Conjugation Results

From 2009.igem.org

(Difference between revisions)
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==Verification and Characterization of Wild R751==
==Verification and Characterization of Wild R751==
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A conjugation test was done using our [[Team:TUDelft/ConjugationProtocol | conjugation protocol]] to determine wild R751 conjugation efficiency.
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'''Donors''': [http://www.cbs.knaw.nl/databases/nccb/search_bac_plas.aspx NCCBNr# 2350] cells with R751 (TRI)<br>
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'''Receivers''': DH5 &alpha; cells with [http://partsregistry.org/Part:BBa_I13522 I13522] (AMP) <br>
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'''Transconjugants''': DH5 &alpha; cells with R751 and [http://partsregistry.org/Part:BBa_I13522 I13522] (TRI+AMP)<br>
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The following table shows the calculated conjugation efficiencies (raw data [https://2009.igem.org/Team:TUDelft/6_August_2009#Calin here]):
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{| border="1" align="center"
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|- align="center"
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| Data Set || Conjugation Efficiency
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|- align="center"
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| 1 ||  0.028
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|- align="center"
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| 2 ||  0.028
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|- align="center"
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| 3 ||  0.025
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|}
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==Verification and Characterization of Conjugation Testing Plasmid 1==
==Verification and Characterization of Conjugation Testing Plasmid 1==
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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.
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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>
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'''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 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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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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{| border="1" align="center"
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|- align="center"
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| Set # || Conjugation Efficiency for Conjugation Testing Plasmid 1 || Conjugation Efficiency for wild R751
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|- align="center"
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| 1 || 0.00177 || 0.0141
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|- align="center"
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| 2 || 0.00224 || 0.0476
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|- align="center"
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| 3|| n/a || 0.0356
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|- align="center"
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|}
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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.
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This test will be redone with R751 &Delta;oriTR cells once they are available.

Revision as of 20:15, 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


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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