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