In this project, we designed a bio-engineered phage for novel citrus greening disease treatment. Through analysis of published transcriptome sequencing dataset and wet lab experiments, we identified promoter P_B488_05770 as a specific and sensitive sensor for LdtR transcriptional factor that is a biomarker of CLas bacteria. The combination of sensor with RinA amplification system constructed a sensing-magnifying system for CLas. Also, we developed an open-source software tool for protein sequence and structural optimization. The antimicrobial peptide that were optimized possesses of higher bacterial lethality. Last but not least, all of our experiments were recorded in details in the publicly shared notebooks, which contents troubleshooting process for references.
MaSAMP was identified as a strong antimicrobial substance for CLas families. We retrieved the amino acid sequences and optimized it with our self-developed software tool ProteinOpti. Amino acid optimization was conducted via substituting amino acids in the hydrophilic face with single of multiple lysine, with aspects including lyticity and total positive hydrophobicity. In the wet lab experiments, the SAMP_6 (BBa_K4587224) was proved to have highest lethality to bacteria. Antimicrobial peptides are of increasing importance in the context that drug resistance is becoming sever. Our optimized AMP with high lethality provides a evolved version of previous MaSAMP, which can be used by later teams. Most importantly, the approaches that we designed and optimized the AMP was also of great reference value to others.
We designed a sensing-respond module can specifically recognize the LdtR marker in CLas bacteria by P_B488_05770, which would be efficiently amplified by RinA amplifier system.
The analysis of the transcriptome data identified a specific sensor for LdtR, which provides a new potential element for citrus greening treatment and prevention. The combination of P_B488_05770 and RinA amplifier system add another piece of evidence for the application of RinA amplifier. The whole module, when put into our engineered phage, is a demonstration of the biosensor usage. Also, the introduction of RinA amplification system added another piece of evidence for application of this amplifier.This module is hopeful to offer a new approach for citrus greening treatment and prevention.
In order to optimize our AMP structurally and in sequences, we developed a protein optimization tool call ProteinOpti. This tool comprehensively considers all factors for sequence improvement. We successfully used it to optimized the AMP sequences and proved them to be efficient. The software tool is open source and shared in our gitlab bank, which is open to be shared across different teams and years.
As one of the most important record of our progresses, the lab notebook recorded all preliminary data, and most importantly, the way and process of how we conquer any difficulties, which is important in wet labs. by sharing our lab notebook, people could easily got the demo approaches and our troubleshooting experiences.