Part:BBa_K2447013:Experience

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Applications of BBa_K2447013

BBA_K2447013 has been characterised using BBa_K2447014 (which is the characterisation construct). Experimental results show that the temperature system is fully functional. We had constructed and characterised ITPG-inducible temperature system (BBa_K2447014) and coupled it to a downstream GFP reporter.

For more detailed experimental results, do visit our [http://2017.igem.org/Team:NUS_Singapore/Experiments experimental page].

BBa_K2447014 is inserted into pBbE2k backbone and subsequently characterised in E. coli 10beta. These cells are grown in LB medium (with kanamycin) at 30 degree Celsius for at least 48 hours before varying concentrations of IPTG from 0 to 500 uM are added. The cells are grown at various temperatures from 30 to 37 degree Celsius for 24 hours before a final GFP reading is taken.

The GFP expression levels correlated well with the temperature to which the cells were subjected to (Figure 1). The highest amount of GFP production were shown at higher temperatures closer to 37 degree Celsius. On the other hand, GFP production is repressed when the temperature is closer to 30 degree Celsius. This is congruent with the [http://www.nature.com/nchembio/journal/v13/n1/abs/nchembio.2233.html?foxtrotcallback=true paper's] literature (Figure 2 and Figure 4) for the thermal sensitive construct which shows much higher GFP expression at temperatures above 36 degree Celsius.

We have reported an optimum condition of 250 uM of IPTG induction (Figure 3) for our temperature sensitive system. In short, our part could prove useful for any group seeking to elucidate gene expression based on temperature as a physical stimulus.

Figure 1: GFP/OD600nm of thermal sensitive construct after 24 hours incubation at various temperatures.

Figure 2: TlpA36 construct as suggested in the [http://www.nature.com/nchembio/journal/v13/n1/abs/nchembio.2233.html?foxtrotcallback=true paper.] Our thermal sensitive construct BBa_K2447014 functioned properly as described in this paper; elucidating much higher GFP expression at temperatures higher than 36 degree Celsius.

Figure 3: When 250 uM of IPTG is added, strong GFP expression is elucidated at high temperatures.

Figure 4: Temperature system is activated and shows a one-fold difference in GFP expression between cultures in 30 and 37 degree Celsius, taken after 6 hours.

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