Team:Buenos Aires/ res basu

From 2013.igem.org

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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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= Response of the construction [[http://parts.igem.org/Part:BBa_K1166000 Bba_K1166000]] under different hypoxia conditions =
= Response of the construction [[http://parts.igem.org/Part:BBa_K1166000 Bba_K1166000]] under different hypoxia conditions =
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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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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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== '''Experiment 1: hypoxia induced by low rate air/medium'''==
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== '''Experiment 1: hypoxia induced by anaerobiosis boxes''' ==
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'''protocol'''
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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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'''Results'''
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== '''Experiment 2: hypoxia induced by anaerobiosis boxes''' ==
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'''protocol'''
'''protocol'''
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== '''Experiment 3: hipoxia induced in a microscope slide sealed''' ==
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== '''Experiment 2: hipoxia induced in a microscope slide sealed''' ==
'''protocol'''
'''protocol'''

Revision as of 00:56, 28 September 2013

Response of the construction http://parts.igem.org/Part:BBa_K1166000 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 2 different assays described below using this strain.

Experiment 1: 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 2: 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