Part:BBa_K2447014

IPTG-inducible temperature-sensitive system with GFP reporter

In the absence of IPTG, LacI repressor will bind to pLac promoter and inhibit further downstream expression. pLac promoter is attached upstream of the TlpA36 protein coding sequence to control the amount of TlpA36 protein expression. When IPTG is present and under high temperatures (above 36 degree Celsius), pTlpA36 will be "ON" and GFP is expressed. Under low temperatures (below 36 degree Celsius), the pTlpA36 will be repressed, which will stop GFP expression.

Usage and Characterisation

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

This part 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.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal NotI site found at 1750
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 1147
  Illegal XhoI site found at 2035
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal AgeI site found at 2338
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI.rc site found at 3303