AW Theeng Mei Jacqueline, BONGSO Nadia Benedicta, CHAN Yuk Pui Alfie, CHEUNG Ming Fung Eric, CHO Haemin, GOTAMA Rinaldi, HAN James, JANG Sang Jin Jason, JOO Minjung, KIM Hye Ji, LEE Jihwan Doyle, M. CUELLAR Raul, MAHANADI Albert, NG Siu Wang Edward, PARK Hojeong Bonnie, SUH Joseph, SULAIMAN Jordy Evan, TANG Chloe, TONG Guillaume, WIDJAJA Angeline Abstract Streptococcus pneumoniae causes health complications such as pneumonia and meningitis, killing around 1.6 million people a year. Although there are existing tools to detect S. pneumoniae, they are not readily accessible in developing countries. So, we designed a new, more accessible diagnostic tool, Pneumosensor. Pneumosensor has a rewired ComCDE signal transduction pathway native to S. pneumoniae that detects autoinducer molecules released specifically by S. pneumoniae. It is also equipped with a new promoter for specific and tight regulation of target gene expression in E. coli. It is important that biosensor has minimum leakage to prevent false alarm. Hence we worked on characterizing riboregulators to increase specificity of expression and minimize leakage that can be applied to our own and other teams’ project. We also took time to characterize PBAD promoter because it is a critical part for the riboregulator system. Detection Module ComD detects CSP and Results will be autophosphorylated by ATP at certain concentration. ComD~P then phosphorylates Figure 1. P and P promoters ComE, following which presence of σ . Scale bar = 5mm. ComE~P induces PcomCDE, and activates comX and comW gene transcription Pneumosensor Background Pneumosensor adopts the quorum sensing pathway in S. pneumoniae to detect its populations. The main advantage of this system is its detection specificity to eliminate possible cross-talk of autoinducer molecules, competence-stimulating peptide (CSP) with native E. coli molecules. celA X This is then incorporated with a highly specific reporting system, whereby σX (ComX) associated with RNAP binds to promoters PcelA and PcomFA containing 8 bp Com-Box sequence specific to σX for activation. Characterization 1. We managed to build and characterize σX CDS (BBa_K1379004), PcelA (BBa_K1379000) and PcomFA (BBa_K1379001). 2. GFP intensities were measured over time to obtain GFP synthesis rates, which then compared to BBa_J23101 as reference promoter to obtain Relative Promoter Units. σX Promoters Module σX associates with RNAP and guides it to the Com-Box sequence on the promoter, initiating transcription of GFP Riboregulator Pneumosensor needs to have minimum leakage to prevent false alarm. Riboregulators can fulfill this requirement. However, a lot of riboregulators in Part Registry are lacking characterization data. Therefore, we characterized those parts so that our team and other teams could use the parts confidently. Riboregulator consists of two components: 1. crRNA is mRNA containing a cis-repressing sequence to sequester the RBS. 2. taRNA contains a sequence complementary to cis-repressive sequence to expose the RBS. comFA activated in the Figure 2. PcelA has 0.53 RPU and PcomFA has 1.21 RPU when paired with σX generator. PBAD Characterization Since PBAD promoter was an essential part of the riboregulator system, we took some time characterizing the promoter. We calculated Relative Promoter Units (RPU) of the promoter in DH10B, BW25113 and DH5α strains to understand how cell strain variability affect the promoter function. All-or-none response was observed for all three cell strains. The levels of RPU, however, were different in the three cell strains. Figure 5. RPU of PBAD promoter in 3 different strains across different arabinose concentrations, using BBa_J23101 as the reference promoter. Human Practice - Start-up Kit _ Start-up Kit is the first comprehensive and handy tool that aims to help iGEM teams, particularly the new iGEMers, in initiating new human practice projects. It includes several items such as: Figure 3. GFP expression measurement for Lock 1 (BBa_J01010) and Key 1 (BBa_J01008) cognate pair. For Lock1 Key1 cognate pair: 1. ~600 fold repression 2. maximum 13 fold activation at 1% arabinose Figure 4. GFP expression measurement for Lock 3 (BBa_J01080) and Key 3 (BBa_J01086) cognate pair. For Lock3 Key3 cognate pair: 1. ~600 fold repression 2. 1.5 fold activation at 1% arabinose; 3 fold activation at 2.5% arabinose 1. Search Engine 2. Handbook 3. Interview with past iGEM Human Practice judges 4. Report Acknowledgement We would like to thank these parties for helping and supporting our teams in completing our projects: 5. Prof. Lam Hon Ming 9. Past iGEM Teams (South University of Science and 11. Wellcome Trust Sanger 1. Office of the Provost, HKUST Institute Technology of China 2014, Tec Monterrey 2013 iGEM, 2. Office of the Dean of Science, HKUST 6. Prof. Tom Richard Manchester 2013, Cornell 2013) 7. Ms. Christine Chiu 3. Professor Patrick Chiu Yat Woo 10. Mr. Kevin Wongso 8. ISF Academy 4. Dr. Don Morrison and all his staffs Figure 6. The porportion of teams conducted Human Practice projects that