Team:Buenos Aires/ res rgfp

From 2013.igem.org

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(→Quantitative measure of HSL production by pArs LuxI construction (Bba_K1106008))
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= AHL production tested with Chromobacterium violaceum =
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=LuxI under Arsenite Inducible Promoter ([http://parts.igem.org/Part:Bba_K1106008 Bba_K1106008]) characterization=
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Chromobacterium violaceum is a bacteria that in presence of certains AHLs turns violet due to the production of violacein.
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== '''AHL production tested with Chromobacterium violaceum''' ==
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Materials:
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'''Materials'''
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Top Agar petri dishes with two layers (agar 0,5% and regular agar 1.5%)
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Chromobacterium violaceum
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TY medium
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LB medium
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Rhizobium leguminosarum as a positive control
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E. coli DH5α harbouring our construct pArs_LuxI
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In order to assess whether the LuxI generator under arsenic promoter synthesizes AHL (a quorum sensing molecule) when induced with arsenite, a top agar petri dish was prepared. A layer of sterile LB-agar 1,5% was poured and on top of it an additional layer of LB-agar 0,5% mixed with a Chromobacterium v. Wells of a volume around 50 ul were performed on the top layer to inoculate different cultures into them.
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*Top Agar petri dishes with two layers (agar 0,5% and regular agar 1.5%)
 +
*Chromobacterium violaceum (CV026 strain)
 +
*TY medium
 +
*LB medium
 +
*Rhizobium leguminosarum as a positive control
 +
*E. coli DH5α harbouring our construct pArs_LuxI
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Four different cultures were grown overnight:
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'''Method'''
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·  A Rhizobium leguminosarum culture, grown at 30 ºC in TY medium, as a positive control.
+
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·  Two E. coli cultures (DH5alpha) harbouring the LuxI enzyme gene of Vibrio fischeri, under the control of an arsenite inducible promoter. One of them was added with 1000 ppb of arsenite.
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·  An E. coli culture without the codifying sequence of LuxI, as a negative control.
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The petri dish, with Chromobacterium v. on the top layer, was inoculated into the wells with samples of 60 ul of each culture and grown overnight at 30ºC. As Chromobacterium v. is a lactone sensitive bacteria, this method allows to detect AHL presence in a easy and very intuitive way by observing the formation of a violet halo around the inoculation wells which results positive.
+
In order to asess if LuxI generator under arsenic sensitive promoter synthesizes AHL (a quorum sensing molecule) when is induced with arsenite, a top agar petri dish was prepared. A layer of sterile LB-agar 1,5% was poured and on top of it an additional layer of LB-agar 0,5% mixed with Chromobacterium v., a bacteria that in presence of certain AHLs turns purple due to the production of violacein. Wells of a volume around 50 ul were performed on the top layer to inoculate different cultures into them.
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Next, the results obtained:  
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Besides, four different cultures were grown overnight:
 +
 +
·  A Rhizobium leguminosarum culture, grown at 30 ºC in TY medium, as a positive control.
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·  Inoculated wells from Rhizobium legumonosarum -the positive control- and  the one from our construct with coding sequence of LuxI that was previously induced with 1000 ppb of arsenite were positive to the presence of AHL, as it’s shown on the photo as a violet halo around the wells.
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·  Two ''E. coli'' cultures (DH5alpha) harbouring the LuxI enzyme gene of Vibrio fischeri, under the control of an arsenite inducible promoter. One of them was added with 1000 ppb of arsenite.
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·  The negative control without a coding sequence of LuxI and the culture from our construct LuxI without added arsenite didn’t show any halo, as it was expected.
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 +
·  An ''E. coli'' culture without the codifying sequence of LuxI, as a negative control.
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= Quantitative measure of HSL production by pArs LuxI construction ([http://parts.igem.org/Part:Bba_K1106008 Bba_K1106008]) =
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Finally, the petri dish, with Chromobacterium v. on the top layer, was inoculated into the wells with samples of 50 ul of each culture and grown overnight at 30ºC. As Chromobacterium v. is a lactone sensitive bacteria, this method allows to detect AHL presence in an easy and very intuitive way by observing the appearance of a violet halo around the inoculation wells which means a positive result.
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'''Protocol'''
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'''Results'''
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four different cultures were grown overnight:
+
[[File:LuxIfede.jpg|center|800px]]
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• An E. coli culture carrying two plasmids that encode an incoherent feedforward system,  
+
· Inoculated wells with Rhizobium leguminosarum, the positive control, and the ''E.coli'' carrying LuxI sequence that was previously induced with 1000 ppb of arsenite, shown positive results.
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designed by Basu et al. ([http://www.ncbi.nlm.nih.gov/pubmed/?term=basu+2004+gfp Basu et al 2004]), which is inducible by Vibrio fischeri’s Lux system.  
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·  The negative control without LuxI coding sequence and the ''E.coli'' carrying LuxI sequence without the addition of arsenite, shown negative results.
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This plasmids were gently provided by Ron Weiss’ lab.
+
== '''Future experiment: quantitative measure of AHL production by pArs LuxI construction''' ==
-
• A Rhizobium leguminosarum culture, grown at 30 ºC in TY medium, as a positive control.
+
'''Method'''
-
• Two E. coli cultures (DH5alpha) harbouring the LuxI enzyme gene of Vibrio fischeri, under the
+
Four different cultures will be grown overnight:
-
control of an arsenite inducible promoter (pArs). One of them was added with 1000 ppb of
+
• An  E. coli culture carrying two plasmids that encode an incoherent feedforward system,designed by Basu et al. ([http://www.ncbi.nlm.nih.gov/pubmed/?term=basu+2004+gfp Basu et al 2004]), which is inducible by Vibrio fischeri’s Lux system. This plasmids were gently provided by Ron Weiss’ lab.
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arsenite.
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[[File:Basu_diseño.jpg|center|600px|]]
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+
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Next day, E. coli culture carrying the incoherent feedforward plasmids was split in three tubes and
+
-
 
+
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centrifuged. The cells were kept the cells, and the supernatant was discarded. Besides, the other
+
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+
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three cultures were centrifuged. The supernatant was kept and the cells were discarded. This  
+
-
 
+
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four conditioned media were used to resuspend the cells carrying Basu’s incoherent feedforward
+
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plasmids.
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Cells were incubated at 37 ºC and every 15 minutes we took an aliquot of each culture, until 105
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minutes after the beginning of the induction.
+
-
 
+
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Afterwards, we measured GFP production at each condition through time.
+
 +
• A Rhizobium leguminosarum culture, grown at 30 ºC in TY medium, as a positive control.
-
'''Results '''
+
• Two E. coli cultures (DH5alpha) harbouring the LuxI enzyme gene of Vibrio fischeri, under the control of an arsenite inducible promoter (pArs). One of them was added with 1000 ppb of arsenite.
-
[[File:Basu_resultados2.jpg|center|600px|]]
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• An E. coli culture (DH5alpha) that doesn´t have the LuxI gene.
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''Positive control''
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[[File:Basu_diseño.jpg|400px|]]
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We observe a high induction of GFP synthesis in cells carrying Basu’s plasmids cultured in medium conditioned by Rhizobium leguminosarum, consistently with what has expected. Despite, probably because the experiment was stopped at an early time, the fall of GFP signal predicted by the model of Basu et al. 2004 in this condition could not be observed.
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The following day, E. coli  culture carrying the incoherent feedforward plasmids will be split in four tubes and centrifuged. We will keep the cells, and discard the supernatant. Besides, the other four cultures will centrifuged. We will keep the supernatant and discard the cells. This four conditioned media will be used to resuspend the cells carrying Basu’s incoherent feedforward plasmids.
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''E. coli harbouring pArs_LuxI conditioned media''
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Cells will be incubated at 37 ºC and every 15 minutes we will take an aliquot of each culture, during 105 minutes after the beginning of the induction.
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Cells cultured in medium conditioned by E. coli pArs_LuxI with 1000 ppb arsenite expressed GFP in a fashion similar to that described by Basu et al. 2004, showing a peak of production around 50 minutes after induction. On the other side, cells cultured in medium conditioned by E. coli pArs_LuxI with 0 ppb arsenite showed a smaller induction and delayed in time, probably due to leaky transcription of the repressed arsenite promoter.
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Afterwards, we will measure GFP production at each condition over the time.
</div>
</div>

Latest revision as of 02:34, 28 September 2013

LuxI under Arsenite Inducible Promoter ([http://parts.igem.org/Part:Bba_K1106008 Bba_K1106008]) characterization

AHL production tested with Chromobacterium violaceum

Materials

  • Top Agar petri dishes with two layers (agar 0,5% and regular agar 1.5%)
  • Chromobacterium violaceum (CV026 strain)
  • TY medium
  • LB medium
  • Rhizobium leguminosarum as a positive control
  • E. coli DH5α harbouring our construct pArs_LuxI

Method

In order to asess if LuxI generator under arsenic sensitive promoter synthesizes AHL (a quorum sensing molecule) when is induced with arsenite, a top agar petri dish was prepared. A layer of sterile LB-agar 1,5% was poured and on top of it an additional layer of LB-agar 0,5% mixed with Chromobacterium v., a bacteria that in presence of certain AHLs turns purple due to the production of violacein. Wells of a volume around 50 ul were performed on the top layer to inoculate different cultures into them.

Besides, four different cultures were grown overnight:

· A Rhizobium leguminosarum culture, grown at 30 ºC in TY medium, as a positive control.

· Two E. coli cultures (DH5alpha) harbouring the LuxI enzyme gene of Vibrio fischeri, under the control of an arsenite inducible promoter. One of them was added with 1000 ppb of arsenite.

· An E. coli culture without the codifying sequence of LuxI, as a negative control.

Finally, the petri dish, with Chromobacterium v. on the top layer, was inoculated into the wells with samples of 50 ul of each culture and grown overnight at 30ºC. As Chromobacterium v. is a lactone sensitive bacteria, this method allows to detect AHL presence in an easy and very intuitive way by observing the appearance of a violet halo around the inoculation wells which means a positive result.

Results

LuxIfede.jpg

· Inoculated wells with Rhizobium leguminosarum, the positive control, and the E.coli carrying LuxI sequence that was previously induced with 1000 ppb of arsenite, shown positive results.

· The negative control without LuxI coding sequence and the E.coli carrying LuxI sequence without the addition of arsenite, shown negative results.

Future experiment: quantitative measure of AHL production by pArs LuxI construction

Method

Four different cultures will be grown overnight:

• An E. coli culture carrying two plasmids that encode an incoherent feedforward system,designed by Basu et al. ([http://www.ncbi.nlm.nih.gov/pubmed/?term=basu+2004+gfp Basu et al 2004]), which is inducible by Vibrio fischeri’s Lux system. This plasmids were gently provided by Ron Weiss’ lab.

• A Rhizobium leguminosarum culture, grown at 30 ºC in TY medium, as a positive control.

• Two E. coli cultures (DH5alpha) harbouring the LuxI enzyme gene of Vibrio fischeri, under the control of an arsenite inducible promoter (pArs). One of them was added with 1000 ppb of arsenite.

• An E. coli culture (DH5alpha) that doesn´t have the LuxI gene.

Basu diseño.jpg

The following day, E. coli culture carrying the incoherent feedforward plasmids will be split in four tubes and centrifuged. We will keep the cells, and discard the supernatant. Besides, the other four cultures will centrifuged. We will keep the supernatant and discard the cells. This four conditioned media will be used to resuspend the cells carrying Basu’s incoherent feedforward plasmids.

Cells will be incubated at 37 ºC and every 15 minutes we will take an aliquot of each culture, during 105 minutes after the beginning of the induction.

Afterwards, we will measure GFP production at each condition over the time.


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