Difference between revisions of "Part:BBa K2447501"
 
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<partinfo>BBa_K2447501 short</partinfo>
 
<partinfo>BBa_K2447501 short</partinfo>
  
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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
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In this construct, the photosensitive bacterial protein is constructively produced. In the presence of blue light, EL222 is activated via dimerisation and is able to bind upstream of the pLuxI promoter, thereby allowing the recruitment of RNA polymerase to activate expression of RFP. In the absence of blue light, EL222 does not dimerise and is unable to bind DNA; hence showing minimal RFP expression.
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[[Image:blue4.jpeg|thumb|left|500px| Figure 1: GFP/OD of thermal sensitive construct after 24 hours incubation at various temperatures]]
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[[Image:blue3.jpeg|thumb|right|500px|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.]]
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[[Image:blue5.png|thumb|center|800px| 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 article.]]]
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====Usage and Characterisation====
 
====Usage and Characterisation====
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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.
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This part is inserted into pBbE8k backbone and subsequently characterized in E. coli 10Beta and MG 1655. These cells are grown in LB medium (with kanamycin) at 37 degrees to reach OD600 of 0.1 before blue light is administered for 7 hours. For every hour, RFP readings were obtained and cross-compared with the control (no blue light).
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[[Image:blue4.jpeg|thumb|center|400px| Figure 2: Incubator-shaker used in the blue light experiment.]]
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[[Image:blue3.jpeg|thumb|center|300px|Figure 3: Blue light being shone on one plate while no light condition was ensured with the covering of a black cloth.]]
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Strong RFP expression was shown from both cell types (under blue-light condition) as compared to the control condition where no light was administered. By the 6th hour onwards, RFP productions had tripled for both cell types when compared to the control condition.
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In short, this construct could prove useful for any group seeking to elucidate gene expression based on blue light as a physical stimulus.
  
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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.
 
  
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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.
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[[Image:blue2.png|thumb|center|800px| Figure 4: MG 1655 under blue light and no light conditions.]]
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[[Image:blue1.png|thumb|center|800px|Figure 5: 10Beta under blue light and no light conditions.]]
  
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[[Image:Temperature1.jpeg|thumb|left|800px| Figure 1: GFP/OD of thermal sensitive construct after 24 hours incubation at various temperatures]]
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<html>
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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.]]
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<div id=WHU-China>
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[[Image:Temperature2.png|thumb|center|800px|Figure 3: When 250 uM of IPTG is added, strong GFP expression is elucidated at high temperatures]]
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</div>
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</html>
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==Characterization by WHU-China 2023==
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We synthesized this composite part and incorporated it into pET-28a. However, in our experiments, it can’t work as expected. There is little difference between the expression of mRFP1 before and after induction in <i>E.coli</i> BL21 with pET-28a-[EL222-mRFP1] (Fig1). After  comparing the sequence with other EL222 protein parts, we found a deletion in the sequence of EL222 from this part (Fig2). In fact, because of the lack of initiation codon, EL222 proteins cannot be produced in bacteria. Based on these facts we found, we designed a new composite part <html><a href="https://parts.igem.org/Part:BBa_K4630301">BBa_K4630301</a></html>, and achieved our own characterization.
  
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<html>
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<figure style="text-align:center;">
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<img src="https://static.igem.wiki/teams/4630/wiki/parts/parts-18.png" width="60%">
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<figcaption><b>Figure 1:</b>Image of mRFP expression in <i>E.coli</i> BL21 with pET-28a-[EL222-mRFP1]</figcaption>
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<img src="https://static.igem.wiki/teams/4630/wiki/parts/parts-19.png" width="100%">
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<figcaption><b>Figure 2:</b>Sequence Alignment of EL222</figcaption>
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</figure>
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</html>
  
 
<!-- Add more about the biology of this part here
 
<!-- Add more about the biology of this part here

Latest revision as of 09:32, 10 October 2023


Blue light activated inducible system with RFP reporter

In this construct, the photosensitive bacterial protein is constructively produced. In the presence of blue light, EL222 is activated via dimerisation and is able to bind upstream of the pLuxI promoter, thereby allowing the recruitment of RNA polymerase to activate expression of RFP. In the absence of blue light, EL222 does not dimerise and is unable to bind DNA; hence showing minimal RFP expression.

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 article.


Usage and Characterisation

This part is inserted into pBbE8k backbone and subsequently characterized in E. coli 10Beta and MG 1655. These cells are grown in LB medium (with kanamycin) at 37 degrees to reach OD600 of 0.1 before blue light is administered for 7 hours. For every hour, RFP readings were obtained and cross-compared with the control (no blue light).

Figure 2: Incubator-shaker used in the blue light experiment.
Figure 3: Blue light being shone on one plate while no light condition was ensured with the covering of a black cloth.


Strong RFP expression was shown from both cell types (under blue-light condition) as compared to the control condition where no light was administered. By the 6th hour onwards, RFP productions had tripled for both cell types when compared to the control condition. In short, this construct could prove useful for any group seeking to elucidate gene expression based on blue light as a physical stimulus.


Figure 4: MG 1655 under blue light and no light conditions.
Figure 5: 10Beta under blue light and no light conditions.

Characterization by WHU-China 2023

We synthesized this composite part and incorporated it into pET-28a. However, in our experiments, it can’t work as expected. There is little difference between the expression of mRFP1 before and after induction in E.coli BL21 with pET-28a-[EL222-mRFP1] (Fig1). After comparing the sequence with other EL222 protein parts, we found a deletion in the sequence of EL222 from this part (Fig2). In fact, because of the lack of initiation codon, EL222 proteins cannot be produced in bacteria. Based on these facts we found, we designed a new composite part BBa_K4630301, and achieved our own characterization.

Figure 1:Image of mRFP expression in E.coli BL21 with pET-28a-[EL222-mRFP1]
Figure 2:Sequence Alignment of EL222

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]