Team:Buenos Aires/ res basu

From 2013.igem.org

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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 Bba_K1166000 under different hypoxia conditions ===
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Five different cultures were grown overnight:
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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 3 different assays described below using this strain. 
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• An  E. coli culture carrying two plasmids that encode an incoherent feedforward system,
 
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designed by Basu et al. (referencia Basu), which is inducible by Vibrio fischeri’s Lux system.
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== Experiment 1: hypoxia induced by low rate air/medium==
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This plasmids were gently provided by Ron Weiss’ lab.
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'''protocol'''
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• A Rhizobium leguminosarum culture, grown at 30 ºC in TY medium, as a positive control.
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In order to provide different oxygen level conditions, E. coli DH5α carrying the plasmid Bba_K1166000 was grown for 24 h in sealed tubes harboring different LB liquid medium and air ratios: 5ml of LB in a 50ml tube (45ml of air), 10ml of LB in a 15ml (5ml of air) tube and 15ml LB in a 15ml tubes(0 ml of air). The first culture was shaken during incubation, while the other two (poor oxygen conditions) were incubated without shaking.
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An aliquot of 1ml was taken from each treatment and was centrifuged. After that, the fluorescence (excitation: 475nm) was measure and normalized by OD600nm.
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• Two E. coli cultures (DH5alpha) harbouring the LuxI enzyme gene of Vibrio fischeri, under the
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'''Results'''
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control of an arsenite inducible promoter (pArs). One of them was added with 1000 ppb of
 
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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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== Experiment 2: hypoxia induced by anaerobiosis boxes ==
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centrifuged. The cells were kept the cells, and the supernatant was discarded. Besides, the other
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'''protocol'''
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three cultures were centrifuged. The supernatant was kept and the cells were discarded. This
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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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four conditioned media were used to resuspend the cells carrying Basu’s incoherent feedforward
 
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plasmids.
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'''results'''
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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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== Experiment 3: hipoxia induced in a microscope slide sealed ==
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Afterwards, we measured GFP production at each condition through time.
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'''protocol'''
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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, 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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'''results'''
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Revision as of 14:06, 24 September 2013

Contents

Response of the construction Bba_K1166000 under different hypoxia conditions

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 3 different assays described below using this strain.


Experiment 1: hypoxia induced by low rate air/medium

protocol

In order to provide different oxygen level conditions, E. coli DH5α carrying the plasmid Bba_K1166000 was grown for 24 h in sealed tubes harboring different LB liquid medium and air ratios: 5ml of LB in a 50ml tube (45ml of air), 10ml of LB in a 15ml (5ml of air) tube and 15ml LB in a 15ml tubes(0 ml of air). The first culture was shaken during incubation, while the other two (poor oxygen conditions) were incubated without shaking. An aliquot of 1ml was taken from each treatment and was centrifuged. After that, the fluorescence (excitation: 475nm) was measure and normalized by OD600nm.

Results


Experiment 2: hypoxia induced by anaerobiosis boxes

protocol

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


Experiment 3: hipoxia induced in a microscope slide sealed

protocol

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


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