Difference between revisions of "Part:BBa K3927002"

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===Description===
 
===Description===
−
This part is an improvement for part BBa_K3927001 (C120-CYC promoter), and aims to improve overall induction fold when induced under blue light while reducing promoter leakiness.
+
3C120-CYC-LacO is the implementation of an abstracted, hypothetical synthetic promoter developed by the NUS iGEM team 2021 for tight, blue light regulated expression in S.cerevisiae.
−
 
+
−
2 additional C120 repeats have been added onto BBa_K3927001 (C120-CYC promoter) upstream of the truncated CYCp core promoter with intent to increase overall induction fold of the promoter in the presence of blue light. A LacO sequence has been added downstream of the TATA box with the goal of providing repression of the inherent promoter leakiness in the absence of blue light.
+
  
 
===Usage===
 
===Usage===
−
 
+
This part is a blue light inducible promoter, therefore requires blue light for this promoter to be induced. Additionally, it requires part <partinfo>BBa_K3570021</partinfo> (NLS-VP16-EL222) and part <partinfo>BBa_K3927006</partinfo> (yeast LacI) to function.
  
 
===Design===
 
===Design===
−
Presented with the dilemma of increasing overall expression while preventing increased leakiness, an abstracted construct of a modular promoter was developed. Modelling for promoter activation kinetics showed that simply increasing the activiy of activation elements is likely to be accompanied by an increase in leakiness. This modular promoter would consist of a core promoter and an upstream activation module that would increase activity in the presence of blue light, similar to the construction of the C120-CYC basic promoter. However, this design includes an additional repression module, that is meant to be repressed in the in darkness to suppress leakiness. In the presence of blue light, this module should be de-repressed, allowing the activated promoter to function as per normal. Thus, it would require two repression modules, one to directly repress the promoter of interest, and a second blue light activated repressor to repress the primary repression module.
+
Our team was motivated to develop a new framework for optogenetic promoters, spurred on by the need to increase inducible expression, but confronted by the dilemma that single layer methods, such as adding additional activation motifs or optimizing the TATA box, were usually accompanied by increased leakiness of the promoter. Taking inspiration from the tightly regulated, yet powerful native promoter GAL1p , it was decided that a combination of conditionally activiating and suppression motifs were required to achieve the desired outcome of both high expresssion and low leakiness.  
−
With this design, it was hoped that strong blue light activation modules with rapid activation kinetics could be developed without the pitfalls of increased leakiness
+
The design that was conceptualized included an artificial upsteam activated module, a core promoter and a reprssion module downstream of the core promoter, drawing from the architecture of native yeast promoters[1].
 +
 
 +
https://2021.igem.org/wiki/images/9/9d/T--NUS_Singapore--results_tab7_1.png
 +
<i>Figure 1: Circuit design for the modular promoter 3C120-CYC-LacO. The abstracted modules include a core promoter, a blue light activated module, and a module for repressing promoter activity</i>
 +
 
 +
In darkness, the repression module prevents leakiness, and in the presence of blue light, a secondary, trans-regulatory repression module represses the primary repressor module, allowing the blue light activated module to power the core promoter. In this way, highly active activation modules could be coupled to the core promoter without the issue of increasining leakiness(Figure 1).
 +
This modularity also meant that activation and repression of the promoter could be controlled separately, allowing for AND gate logic, where represson module could be linked to an alternative response(Figure 2).
 +
 
 +
https://static.igem.org/mediawiki/parts/5/5d/T--NUS_Singapore--3C120-CYC-LacO_architecture.png
 +
 
 +
<i>Figure 2: 3C120-CYC-LacO can either be used to tighten expression of a single blue light input by linking LacI to a secondary, blue light repressed module, or can be coupled to alternative</i>
 +
 
 +
To implement this design, the NUS iGEM team 2021 decided to further improve the optogenetic system in part BBa_K3570005 designed by the Toulouse iGEM team 2020. The part depends on the expression of an NLS-VP16-EL222 fusion transcription factos, which dimerizes in blue light and binds to C120 repeats[2], activating a core CYC1 promoter element in close proximity(Figure 1). Thus, our chosen activation module a 3x repeat of the C120 motif.
 +
For the primary repression module, a Lac operon sequence was chosen, at it has been demonstrated to be a functional repressor of synthetic promoters in yeast[3]. This was inserted downstream of the TATA box in the core CYC1 promoter element.
 +
 
 +
https://static.igem.org/mediawiki/parts/b/b0/T--NUS_Singapore--EL222_blue_light_activated_transcription_system.png
 +
<i>Figure 3: EL222 blue light activated transcription system.</i>
 +
 
 +
For the primary repression module, a Lac operon sequence was chosen, at it has been demonstrated to be a functional repressor of synthetic promoters in yeast[3]. This was inserted downstream of the TATA box in the core CYC1 promoter element.
  
 
===Characterization===
 
===Characterization===
−
This part was characterized by placing the part upstream of a mKO reporter gene. The overall performance of the composite part was quantified based on the levels of mKO produced.
+
<b> Characterization of activation module </b>
 +
 
 +
https://static.igem.org/mediawiki/parts/6/67/T--NUS_Singapore--3C120-CYC-LacO_activity_without_LacI_module.png
 +
 
 +
<i>Figure 4: RFU of 3C120-CYC-LacO promoter controlling fluorescent protein mKO with concurrent EL222 expression, in either dark or blue light for 6 hours, compared to wildtype yeast</i>
 +
 
 +
Figure 4 demonstrates 3C120-CYC-LacO is successfully activated in blue light when housed in S.cerevisiae constitutively expressing NLS-VP16-EL222, demonstrating increased mKO expression compared to wildtype yeast when cultured in blue light.
 +
 
 +
https://static.igem.org/mediawiki/parts/thumb/2/2f/T--NUS_Singapore--Induction_of_3C120-CYC-LacO_over_time.png/800px-T--NUS_Singapore--Induction_of_3C120-CYC-LacO_over_time.png
 +
 
 +
<i>Figure 5: Expression of mKO of synthetic promoter in darkness, 50% light and 100% blue light, 50% light was carried out using half hour duty cycles of blue light.</i>
 +
 
 +
Figure 5 represents the expression values in RFU over time for 3C120-CYC-LacO in S.cerevisiae constitutively expressing NLS-VP16-EL222 in either darkness, full blue light or half hour on-half hour off cycles of blue light. 50% cycles of blue light showed a more gradual response than 100% blue light, demonstrating the ability of 3C120-CYC-LacO to be activated in a dose-dependent manner.
 +
 
 +
https://static.igem.org/mediawiki/parts/thumb/f/fa/T--NUS_Singapore--OD_of_pGL3CM-H_over_time.png/800px-T--NUS_Singapore--OD_of_pGL3CM-H_over_time.png
 +
 
 +
<i>Figure 6: OD600 of yeast harbouring the synthetic promoter in darkness, 50% light and 100% blue light, 50% light was carried out using half hour duty cycles of blue light.</i>
 +
 
 +
Figure 6 shows the growth curve of S.cerevisiae constitutively expressing NLS-VP16-EL222 and harbouring a plasmid containing 3C120-CYC-LacO in in either darkness, full blue light or half hour on-half hour off cycles of blue light. Data represents minimal difference in cells with an active promoter, 50% active promoter(dose dependent activity is established by Figure 5) or inactive promoter in terms of growth, and thus this part does not directly impose a metabolic burden on the cell.
 +
 
 +
<b> Characterization of cis-repression module </b>
 +
 
 +
https://static.igem.org/mediawiki/parts/thumb/9/95/T--NUS_Singapore--Blue_Light_AND_NOT_LacI_Gate.png/800px-T--NUS_Singapore--Blue_Light_AND_NOT_LacI_Gate.png
 +
 
 +
<i> Figure 7: Induction of 3C120-CYC-LacO when accompanied by both constitutive EL222 and LacI expression, cultured in either dark, blue light with and without 5mM IPTG added. </i>
 +
 
 +
Cells harbouring a plasmid containing 3C120-CYC-LacO as well as constitutively expressing both an  NLS-VP16-EL222 activation factor and LacI repressor protein were used to characterize the LacO repression module(Figure 7). Cells grown in either darkness or blue light showed minimal expression, demonstrating successful repression by the LacI protein being expressed. When 5mM IPTG was added in the darkness, LacI was allosterically inhibited, relieving repression of the 3C120-CYC-LacO promoter and restoring basal levels of leakiness, which conversely highlighted the ability of LacI to supress this leakiness. Finally, in both blue light and 5mM IPTG, the promoter was fully active, demonstrating the ability of the promoter to carry out AND gated logic for either coupling blue light to a secondary input linked to the expression of LacI, or reducing leakiness of the blue light induction.
 +
 
 +
<b> Significance </b>
 +
 
 +
This promoter demonstrates the ability to construct a modular promoter by combining sequences regulated by trans-regulatory factors. Optogenetics is infamous for high leakiness, and this promoter represents a framework that could be used to counteract that issue, as well as providing the opportunity for the construction of combinatorial logic with other secondary outputs through the conditional expression/repression of a trans-LacI element. We also hope that this framework will inspire others to turn to modular promoters as a means to improve their circuits.
 +
 
 +
===References===
 +
 
 +
1. Tang, H., Wu, Y., Deng, J., Chen, N., Zheng, Z., Wei, Y., Luo, X., & Keasling, J. D. (2020). Promoter Architecture and Promoter Engineering in Saccharomyces cerevisiae. Metabolites, 10(8), 320. https://doi.org/10.3390/metabo10080320
  
−
A DNA fragment containing the C120-CYC promoter, as well as a constitutive expression cassette for NLS-VP16-EL222 was ordered from IDT, and Gibson assembly was used to assemble it into a plasmid with the mKO orange fluorescent protein terminated by LSC2 terminator, producing the plasmid pC120-mKO-EL-U. The plasmid was then transformed into BY4741 to test for EL222 mediated blue light induction of mKO.
+
2. Benzinger D, Khammash M. Pulsatile inputs achieve tunable attenuation of gene expression variability and graded multi-gene regulation. Nat Commun. 2018 Aug 30;9(1):3521. doi: 10.1038/s41467-018-05882-2. PMID: 30166548; PMCID: PMC6117348.
  
−
pC120-mKO-EL-U(BY4741) was compared to the wildtype BY4741 as a negative control. Cells were cultured overnight, and the next day two cultures inoculated in 25ml YNB-URA media to OD600~1.2, and then cultured in a shaking incubator at 30 degrees Celsius, 220rpm in either blue light or darkness, with wildtype BY4741 undergoing an identical protocol. After 6 hours, the cells were washed and the level of mKO fluorescence(Ex:515, Em:560) was measured and normalized to the OD of the culture to ascertain the level of mKO expression under the C120-CYC promoter.
+
3. Pothoulakis G, Ellis T. Synthetic gene regulation for independent external induction of the Saccharomyces cerevisiae pseudohyphal growth phenotype. Commun Biol. 2018 Jan 22;1:7. doi: 10.1038/s42003-017-0008-0. PMID: 30271894; PMCID
  
−
Other than the absolute induction fold, it was also important to characterize the effect of dose depended activation as well as the possible metabolic burden that the circuit may impose. As such, the experiment was replicated with the same induction protocol, but instead of an endpoint measurement the OD600 and mKO expression was measured hourly to plot the expression and growth curve in darkness, 100% blue light or using a 50%, half-hour-on half hour-off cycle.
 
  
 
<!-- Uncomment this to enable Functional Parameter display  
 
<!-- Uncomment this to enable Functional Parameter display  

Latest revision as of 14:11, 18 October 2021


3C120-CYC-LacO

This part encodes for a truncated CYCp core promoter with three C120 repeats replacing the native upstream activating sequence, and a lacO sequence downstream of the TATA box.

Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal XbaI site found at 203
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 185
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal XbaI site found at 203
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal XbaI site found at 203
  • 1000
    COMPATIBLE WITH RFC[1000]

Description

3C120-CYC-LacO is the implementation of an abstracted, hypothetical synthetic promoter developed by the NUS iGEM team 2021 for tight, blue light regulated expression in S.cerevisiae.

Usage

This part is a blue light inducible promoter, therefore requires blue light for this promoter to be induced. Additionally, it requires part BBa_K3570021 (NLS-VP16-EL222) and part BBa_K3927006 (yeast LacI) to function.

Design

Our team was motivated to develop a new framework for optogenetic promoters, spurred on by the need to increase inducible expression, but confronted by the dilemma that single layer methods, such as adding additional activation motifs or optimizing the TATA box, were usually accompanied by increased leakiness of the promoter. Taking inspiration from the tightly regulated, yet powerful native promoter GAL1p , it was decided that a combination of conditionally activiating and suppression motifs were required to achieve the desired outcome of both high expresssion and low leakiness. The design that was conceptualized included an artificial upsteam activated module, a core promoter and a reprssion module downstream of the core promoter, drawing from the architecture of native yeast promoters[1].

T--NUS_Singapore--results_tab7_1.png Figure 1: Circuit design for the modular promoter 3C120-CYC-LacO. The abstracted modules include a core promoter, a blue light activated module, and a module for repressing promoter activity

In darkness, the repression module prevents leakiness, and in the presence of blue light, a secondary, trans-regulatory repression module represses the primary repressor module, allowing the blue light activated module to power the core promoter. In this way, highly active activation modules could be coupled to the core promoter without the issue of increasining leakiness(Figure 1). This modularity also meant that activation and repression of the promoter could be controlled separately, allowing for AND gate logic, where represson module could be linked to an alternative response(Figure 2).

T--NUS_Singapore--3C120-CYC-LacO_architecture.png

Figure 2: 3C120-CYC-LacO can either be used to tighten expression of a single blue light input by linking LacI to a secondary, blue light repressed module, or can be coupled to alternative

To implement this design, the NUS iGEM team 2021 decided to further improve the optogenetic system in part BBa_K3570005 designed by the Toulouse iGEM team 2020. The part depends on the expression of an NLS-VP16-EL222 fusion transcription factos, which dimerizes in blue light and binds to C120 repeats[2], activating a core CYC1 promoter element in close proximity(Figure 1). Thus, our chosen activation module a 3x repeat of the C120 motif. For the primary repression module, a Lac operon sequence was chosen, at it has been demonstrated to be a functional repressor of synthetic promoters in yeast[3]. This was inserted downstream of the TATA box in the core CYC1 promoter element.

T--NUS_Singapore--EL222_blue_light_activated_transcription_system.png Figure 3: EL222 blue light activated transcription system.

For the primary repression module, a Lac operon sequence was chosen, at it has been demonstrated to be a functional repressor of synthetic promoters in yeast[3]. This was inserted downstream of the TATA box in the core CYC1 promoter element.

Characterization

Characterization of activation module

T--NUS_Singapore--3C120-CYC-LacO_activity_without_LacI_module.png

Figure 4: RFU of 3C120-CYC-LacO promoter controlling fluorescent protein mKO with concurrent EL222 expression, in either dark or blue light for 6 hours, compared to wildtype yeast

Figure 4 demonstrates 3C120-CYC-LacO is successfully activated in blue light when housed in S.cerevisiae constitutively expressing NLS-VP16-EL222, demonstrating increased mKO expression compared to wildtype yeast when cultured in blue light.

800px-T--NUS_Singapore--Induction_of_3C120-CYC-LacO_over_time.png

Figure 5: Expression of mKO of synthetic promoter in darkness, 50% light and 100% blue light, 50% light was carried out using half hour duty cycles of blue light.

Figure 5 represents the expression values in RFU over time for 3C120-CYC-LacO in S.cerevisiae constitutively expressing NLS-VP16-EL222 in either darkness, full blue light or half hour on-half hour off cycles of blue light. 50% cycles of blue light showed a more gradual response than 100% blue light, demonstrating the ability of 3C120-CYC-LacO to be activated in a dose-dependent manner.

800px-T--NUS_Singapore--OD_of_pGL3CM-H_over_time.png

Figure 6: OD600 of yeast harbouring the synthetic promoter in darkness, 50% light and 100% blue light, 50% light was carried out using half hour duty cycles of blue light.

Figure 6 shows the growth curve of S.cerevisiae constitutively expressing NLS-VP16-EL222 and harbouring a plasmid containing 3C120-CYC-LacO in in either darkness, full blue light or half hour on-half hour off cycles of blue light. Data represents minimal difference in cells with an active promoter, 50% active promoter(dose dependent activity is established by Figure 5) or inactive promoter in terms of growth, and thus this part does not directly impose a metabolic burden on the cell.

Characterization of cis-repression module

800px-T--NUS_Singapore--Blue_Light_AND_NOT_LacI_Gate.png

Figure 7: Induction of 3C120-CYC-LacO when accompanied by both constitutive EL222 and LacI expression, cultured in either dark, blue light with and without 5mM IPTG added.

Cells harbouring a plasmid containing 3C120-CYC-LacO as well as constitutively expressing both an NLS-VP16-EL222 activation factor and LacI repressor protein were used to characterize the LacO repression module(Figure 7). Cells grown in either darkness or blue light showed minimal expression, demonstrating successful repression by the LacI protein being expressed. When 5mM IPTG was added in the darkness, LacI was allosterically inhibited, relieving repression of the 3C120-CYC-LacO promoter and restoring basal levels of leakiness, which conversely highlighted the ability of LacI to supress this leakiness. Finally, in both blue light and 5mM IPTG, the promoter was fully active, demonstrating the ability of the promoter to carry out AND gated logic for either coupling blue light to a secondary input linked to the expression of LacI, or reducing leakiness of the blue light induction.

Significance

This promoter demonstrates the ability to construct a modular promoter by combining sequences regulated by trans-regulatory factors. Optogenetics is infamous for high leakiness, and this promoter represents a framework that could be used to counteract that issue, as well as providing the opportunity for the construction of combinatorial logic with other secondary outputs through the conditional expression/repression of a trans-LacI element. We also hope that this framework will inspire others to turn to modular promoters as a means to improve their circuits.

References

1. Tang, H., Wu, Y., Deng, J., Chen, N., Zheng, Z., Wei, Y., Luo, X., & Keasling, J. D. (2020). Promoter Architecture and Promoter Engineering in Saccharomyces cerevisiae. Metabolites, 10(8), 320. https://doi.org/10.3390/metabo10080320

2. Benzinger D, Khammash M. Pulsatile inputs achieve tunable attenuation of gene expression variability and graded multi-gene regulation. Nat Commun. 2018 Aug 30;9(1):3521. doi: 10.1038/s41467-018-05882-2. PMID: 30166548; PMCID: PMC6117348.

3. Pothoulakis G, Ellis T. Synthetic gene regulation for independent external induction of the Saccharomyces cerevisiae pseudohyphal growth phenotype. Commun Biol. 2018 Jan 22;1:7. doi: 10.1038/s42003-017-0008-0. PMID: 30271894; PMCID