Part:BBa_K3927006
yeLacI
LacI codon optimized for expression in S.cerevisiae
Sequence and Features
- 10INCOMPATIBLE WITH RFC[10]Illegal XbaI site found at 253
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 1044
- 21COMPATIBLE WITH RFC[21]
- 23INCOMPATIBLE WITH RFC[23]Illegal XbaI site found at 253
- 25INCOMPATIBLE WITH RFC[25]Illegal XbaI site found at 253
Illegal NgoMIV site found at 595 - 1000COMPATIBLE WITH RFC[1000]
Description
LacI is a bacterial repressor commonly used in genetic circuits[1]. It has also been shown to be able to repress it’s cognate LacO sequence when expressed in S.cerevisiae[2]. The NUS iGEM 2021 team thus added a yeast optimized LacI to be able to control synthetic promoters in a repressible manner.
Usage
LacI can be used as a transregulatory element for promoters containing the Lac operon sequence downstream of the TATA box
Characterisation
To test the functionality of this part, the synthetic promoter 3C120-CYC-LacO was used, which contained a LacO sequence downstream of it’s TATA box. A plasmid constitutively expressing LacI was co-transformed with a plasmid containing 3C120-CYC-LacO upstream of a reporter gene mKO into S.cerevisiae strain BY4741 containing necessary transcriptional machinery to activate 3C120-CYC-LacO in blue light.
The yeast were then cultured for 48 hours in either dark or light, with and without 5mM IPTG to simulate repression of LacI (Figure 1).
Promoter clearly shows repression in both light and darkness, when IPTG was added without blue light, the promoter showed increased leakiness, and when both blue light and IPTG was present promoter was fully activated.
Significance
LacI demonstrates the ability to repress promoters in S.cerevisiae, which may be especially helpful in reducing unwanted expression of highly leaky constructs such as optogenetic circuits. Alternatively, LacI can be used in conjunction with LacO containing promoters to implement AND-gate logic.
References
1. Savageau M. A. (2011). Design of the lac gene circuit revisited. Mathematical biosciences, 231(1), 19–38. https://doi.org/10.1016/j.mbs.2011.03.008
2. 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
| None |