Part:BBa_K3927002
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
- 10INCOMPATIBLE WITH RFC[10]Illegal XbaI site found at 203
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 185
- 21COMPATIBLE WITH RFC[21]
- 23INCOMPATIBLE WITH RFC[23]Illegal XbaI site found at 203
- 25INCOMPATIBLE WITH RFC[25]Illegal XbaI site found at 203
- 1000COMPATIBLE 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].
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).
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.
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
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.
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.
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
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
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