Team:Hong Kong HKUST/pLac
lacp Module
The lacp module consists of a Lac repressible promoter upstream of the CDSs of TetR and PhlF, which are then followed by the reporter gene, sfGFP. When lacp is activated, the two repressor proteins, TetR and PhlF will be expressed along with the reporter gene, sfGFP. This results in the repression of the two other modules in our system, phlFp and tetp.
Figure 1. lacp module
Promoter, Repressor and Inducer Specification
- Promoter name: lacp (Lac repressible promoter)
- Length: 200 base pairs
- Strength with:
- Weak RBS (BBa_B0032): 290.27-195.5 (+17.7) RPU
- Medium RBS (BBa_B0030): 11.347-9456(+487) RPU
- Strong RBS (BBa_B0034): 1.126-7648 (+152) RPU
In gram negative bacteria, LacI represses lacp which regulates the binding of RNA polymerase and inhibits transcription by binding to the lac operon.
- Repressor name: LacI (LacI repressor)
- Part Length: 1153 base pairs
- Structure: Homo-tetramer
- Binding affinity: To lacp, very tight, 60.145M
The LacI repressor is an allosteric protein exists in 2 states: silent and active. In the absence of inducers, the active state of LacI binds to the lac operator sites of the lacp promoter and interferes the binding the RNA polymerase, thus inhibiting transcription. In the presence of inducers, however, LacI undergoes allosteric rearrangement and switches to its silent state, resulting in its release from the promoter, thus allowing transcription to take place.
- Inducer name: IPTG (Isopropyl β-D-1-thiogalactopyranoside)
- Effective Concentration : 20 ng/ml
In our project, Isopropyl β-D-1-thiogalactopyranoside (IPTG) was used to induce lacp driven expressions. IPTG is a lactose metabolite, lacp’s native inducer, which has shown stronger binding affinity to LacI. On top of that, it cannot be metabolized by Escherichia coli so it concentration remains constant during the experiment, making it a more suitable inducer for our Tristable Switch system.
Mechanism of lacp - LacI - IPTG Interaction
Figure 2. Behaviour of LacI in the presence/absence of IPTG
In the absence of IPTG, LacI would bind to lacp, inhibiting transcription and thus bringing sfGFP expression to a halt. In the presence of IPTG, however, LacI would lose the ability to bind to lacp, allowing transcription to proceed, resulting sfGFP expression.
References
1. Aleksandrov, A., Schuldt, L., Hinrichs, W., & Simonson, T. (2008). Tet Repressor Induction by Tetracycline: A Molecular Dynamics, Continuum Electrostatics, and Crystallographic Study. Journal Of Molecular Biology, 378(4), 898-912. http://dx.doi.org/10.1016/j.jmb.2008.03.022
2. Chen and Wang. (2014). Synthetic Gene Network: Modeling, Analysis and Robust Design Methods.
3. Gossen, M. & Bujard, H. (1993). Anhydrotetracycline, a novel effector for tetracycline controlled gene expression systems in eukaryotic cells. Nucleic Acids Research, 21(18), 4411-4412. http://dx.doi.org/10.1093/nar/21.18.4411
4. Lederer, T., Takahashi, M., & Hillen, W. (1995). Thermodynamic Analysis of Tetracycline-Mediated Induction of Tet Repressor by a Quantitative Methylation Protection Assay. Analytical Biochemistry, 232(2), 190-196. http://dx.doi.org/10.1006/abio.1995.0006
5. Oliva, B., Gordon, G., McNicholas, P., Ellestad, G., & Chopra, I. (1992). Evidence that tetracycline analogs whose primary target is not the bacterial ribosome cause lysis of Escherichia coli. Antimicrobial Agents And Chemotherapy, 36(5), 913-919. http://dx.doi.org/10.1128/aac.36.5.913
