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), pTlpa36 will be constitutively and eliciting downstream GFP expression. Under low temperatures (below 36 degree), the pTlpa36 will be repressed, and drastically reducing GFP expression

Usage and Characterisation

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

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

At 250uM of IPTG (figure 3), we have reported optimum condition 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/OD 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 had functioned properly as described in this paper; elucidating much higher GFP expression at temperatures higher than 36 degree.

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

Figure 4: Temperature system is activated and indicated by the one-fold increase in GFP expression when the temperature is at 37 degree. At 30 degree, tlpa36 proteins dimerises and attach to the promoter tlpa36, thereby reducing downstream expression of GFP.

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