Difference between revisions of "Part:BBa K2447501"

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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
 
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
[[Image:blue4.jpeg|thumb|center|400px| Figure 1: Incubator-shaker used in the blue light experiment.]]
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[[Image:blue5.jpeg|thumb|center|400px| Figure 1: Under blue-light condition, EL222 is recruited upstream of the RNA polymerase binding region and recruits the polymerase for downstream expression of RFP. Figure is obtained from this [https://www.ncbi.nlm.nih.gov/pubmed/27353329 paper.]]]
[[Image:blue3.jpeg|thumb|center|300px|Figure 2: Blue light being shone on one plate while no light condition was stimulated with the covering of a black cloth.]]
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====Usage and Characterisation====
 
====Usage and Characterisation====
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[[Image:Temperature3.png|thumb|right|400px|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.]]
 
[[Image:Temperature3.png|thumb|right|400px|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.]]
 
[[Image:Temperature2.png|thumb|center|800px|Figure 3: When 250 uM of IPTG is added, strong GFP expression is elucidated at high temperatures]]
 
[[Image:Temperature2.png|thumb|center|800px|Figure 3: When 250 uM of IPTG is added, strong GFP expression is elucidated at high temperatures]]
 +
[[Image:blue4.jpeg|thumb|center|400px| Figure 1: Incubator-shaker used in the blue light experiment.]]
 +
[[Image:blue3.jpeg|thumb|center|300px|Figure 2: Blue light being shone on one plate while no light condition was stimulated with the covering of a black cloth.]]
  
  

Revision as of 14:53, 18 October 2017


Blue light activated inducible system with RFP 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

File:Blue5.jpeg
Figure 1: Under blue-light condition, EL222 is recruited upstream of the RNA polymerase binding region and recruits the polymerase for downstream expression of RFP. Figure is obtained from this paper.


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) 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 1: Incubator-shaker used in the blue light experiment.
Figure 2: Blue light being shone on one plate while no light condition was stimulated with the covering of a black cloth.


Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 7
    Illegal NheI site found at 30
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal XhoI site found at 527
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 97
    Illegal AgeI site found at 1473
    Illegal AgeI site found at 1585
  • 1000
    COMPATIBLE WITH RFC[1000]