Authors：Huang Tianze, Zhou Manxuan, Chen Xinyi, Yan Qi, Shen Yunhao, Wu Yan, Zhou Liqun, Mao Zeping, Yao Liang, Han Jianing, Zhou Siyan, Wang Xuan, Wang Tingxuan, Zheng Kaiyu, Ge Xiaofei, Li Yibo, Hao Yuejun.
Instructors：Dr. Chen Guoqiang, Dr. Liu Dong, Dr. Sun Zhirong, Dr. Li Peng
Keyword: Positive Feedback Quorum Sensing AHL
Abstract
Positive feedback circuits can be an approach for synthetic circuits to realize complex functions, such as logic gates and toggle switchs. Here we designed a quorum sensing positive feedback loop to achieve high signal output. To avoid leakage, we introduced the CRISPRi system to subdue the positive feedback loop. We named it NEON system. It is a wide-dynamic-range,fine-tuned quorum sensing positive feedback circuit.
Methods
- Molecular Cloning
- Spectrophotometry
- Flow Cytometry
We designed a series of lux pR mutants to evaluate their leakage level and reaction to AHL stimulation.
Hypersensitive promoter: lux pR-HS (BBa_K2558001)
We used Anderson promotor J23100, J23110 and J23114 to design three constitutive lacI generators of different intensities.The correlation between lacI dosage and IPTG induction efficiency
was clarified.
We observed how IPTG induced CRISPRi process proceeded under different levels of lacI expression.
Figure.1. lux pR Mutation Analysis
Figure. 2. lacI Dosage & IPTG Induction
Figure. 3. IPTG induced CRISPRi
Model
Figure. 4. The influence of transcription efficiency of lux pR on Neon and Safety Catch plasmids on the behavior of the system.
Figure.5. System stablity influenced by lux pR leakage on Safety Catch plasmid.（left） & NEON simulation with various initial AHL concentration.（right）
Results
Figure.6. Leakage and 10-10 mM AHL stimulation of
lux pR promotor and its mutations.
Figure.7.Leakage and AHL stimulation of lux pR Mutant 5 and Mutant 10.
Figure.8. The effect of varied lacI-LVA (BBa_C0011) promotor strength on IPTG induction of Tac promotor (BBa_K2558004).
Conclusion
In conclusion, we establish a controllable feedback system. It could be employed in various synthetic biology projects and could well improve the performance of prokaryotic expression and signal transduction systems.
