Team:Buenos Aires/ res basu

From 2013.igem.org

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(Created page with "<div id="external"> = Basu = Quantitative measure of HSL production by pArs LuxI construction ([http://parts.igem.org/Part:Bba_K1106008 Bba_K1106008]) Five different cultures w...")
 
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= Basu =
 
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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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= Response of the construction [[http://parts.igem.org/Part:BBa_K1166000 Bba_K1166000]] under different hypoxia conditions =
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Five different cultures were grown overnight:
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'''Objective'''
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• An  E. coli culture carrying two plasmids that encode an incoherent feedforward system,
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We aimed to characterize the part sent by de Monterrey Team which is a GFP under a hypoxia inducible promoter, cloned in a replicative plasmid (Bba_K1166000). For that purpose we transformed E. coli DH5α with plamid Bba_K1166000 and conducted 2 different assays described below using this strain.
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designed by Basu et al. (referencia Basu), which is inducible by Vibrio fischeri’s Lux system.
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This plasmids were gently provided by Ron Weiss’ lab.
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== '''Experiment 1: hypoxia induced by anaerobiosis boxes''' ==
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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
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'''Method'''
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control of an arsenite inducible promoter (pArs). One of them was added with 1000 ppb of
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A 5ml culture of E. coli DH5α carrying the plasmid Bba_K1166000 in LB medium was grown for 16 h in aerobiosis, 1 ml was centrifuged and the pellet was kept frozen in order to use it as fluorescence control without induction. The rest of the culture was incubated for 20 h under anaerobic and microanaerobic conditions using hermetic boxes and GENbox aner and GENbox microanaer systems (bioMérieux), respectively, without shacking at 37°C. GENbox aner and GENbox microanaer systems are based on a chemical compound that traps oxygen.
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One ml of each condition was centrifuged and the pellets frozen. Finally, fluorescence (excitation: 475 nm) was measure and normalized by OD600nm. Both treatments were performed in duplicate.
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arsenite.
 
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Next day, E. coli  culture carrying the incoherent feedforward plasmids was split in three tubes and
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'''Results'''
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centrifuged. The cells were kept the cells, and the supernatant was discarded. Besides, the other
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[[File:Hernanmexpng.PNG|center|600px]]
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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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== '''Experiment 2: hipoxia induced in a microscope slide sealed''' ==
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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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'''Method'''
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minutes after the beginning of the induction.
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An E. coli DH5α carrying the plasmid Bba_K1166000  was grown under aerobic conditions overnight. Then, 10ul of this culture was placed in a microscope slide, covered with a coverslip and sealed with nail enamel (similar as usually performed to induce hypoxia in plant cells).
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Afterwards, we measured GFP production at each condition through time.
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Afterwards, pictures of bacteria were taken in a fluorescence microscope every 15 min for 1 h. The first picture was taken immediately after sealing. Every picture was taken using the same exposition time (1/5 sec). Unfortunately, a lot of bleaching was observed. To avoid this, the field was changed before taking each photo. From 0 min, bacteria exhibited green fluorescence, however no significant induction can be observed in the time points analyzed (15, 30, 45 and 60 min).  
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{| class="wikitable" style="background-color:#fff; margin:auto; text-align:center;"
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|+Results
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|[[File:Carlotto1.JPG|center|600px]]
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Fluorescence microscope picture 0 minutes after sealing
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[[File:Carlotto15min.JPG|center|600px]]
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Fluorescence microscope picture 15 minutes after sealing
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|[[File:Carlotto30min.JPG|center|600px]]
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Fluorescence microscope picture 30 minutes after sealing
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|[[File:Carlotto45min.JPG|center|600px]]
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Fluorescence microscope picture 45 minutes after sealing
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[[File:Carlotto60min.JPG|center|600px]]
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Fluorescence microscope picture 60 minutes after sealing
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== '''Overall conclusions''' ==
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Based on the results obtained, GFP is beeing produced. However, FNR-HIP1 promoter doesn't seem to respond to the oxygen concentration in growth medium. The fluorescence microscopy assay shows no variation between different oxygen conditions. Moreover, in the other experiment, GFP seems to be produced in higher amounts when bacteria were grown in the presence of oxygen.
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Latest revision as of 19:06, 26 October 2013

Contents

Response of the construction http://parts.igem.org/Part:BBa_K1166000 Bba_K1166000 under different hypoxia conditions

Objective

We aimed to characterize the part sent by de Monterrey Team which is a GFP under a hypoxia inducible promoter, cloned in a replicative plasmid (Bba_K1166000). For that purpose we transformed E. coli DH5α with plamid Bba_K1166000 and conducted 2 different assays described below using this strain.



Experiment 1: hypoxia induced by anaerobiosis boxes

Method

A 5ml culture of E. coli DH5α carrying the plasmid Bba_K1166000 in LB medium was grown for 16 h in aerobiosis, 1 ml was centrifuged and the pellet was kept frozen in order to use it as fluorescence control without induction. The rest of the culture was incubated for 20 h under anaerobic and microanaerobic conditions using hermetic boxes and GENbox aner and GENbox microanaer systems (bioMérieux), respectively, without shacking at 37°C. GENbox aner and GENbox microanaer systems are based on a chemical compound that traps oxygen. One ml of each condition was centrifuged and the pellets frozen. Finally, fluorescence (excitation: 475 nm) was measure and normalized by OD600nm. Both treatments were performed in duplicate.


Results

Hernanmexpng.PNG



Experiment 2: hipoxia induced in a microscope slide sealed

Method

An E. coli DH5α carrying the plasmid Bba_K1166000 was grown under aerobic conditions overnight. Then, 10ul of this culture was placed in a microscope slide, covered with a coverslip and sealed with nail enamel (similar as usually performed to induce hypoxia in plant cells).

Afterwards, pictures of bacteria were taken in a fluorescence microscope every 15 min for 1 h. The first picture was taken immediately after sealing. Every picture was taken using the same exposition time (1/5 sec). Unfortunately, a lot of bleaching was observed. To avoid this, the field was changed before taking each photo. From 0 min, bacteria exhibited green fluorescence, however no significant induction can be observed in the time points analyzed (15, 30, 45 and 60 min).


Results
Carlotto1.JPG

Fluorescence microscope picture 0 minutes after sealing

Carlotto15min.JPG

Fluorescence microscope picture 15 minutes after sealing

Carlotto30min.JPG

Fluorescence microscope picture 30 minutes after sealing

Carlotto45min.JPG

Fluorescence microscope picture 45 minutes after sealing

Carlotto60min.JPG

Fluorescence microscope picture 60 minutes after sealing


Overall conclusions

Based on the results obtained, GFP is beeing produced. However, FNR-HIP1 promoter doesn't seem to respond to the oxygen concentration in growth medium. The fluorescence microscopy assay shows no variation between different oxygen conditions. Moreover, in the other experiment, GFP seems to be produced in higher amounts when bacteria were grown in the presence of oxygen.

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