Hong Kong HKUST Championship Poster

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