"
Team:NYMU-Taipei/project/2c
From 2014.igem.org
- The part aims to combine the two S. mutans killing system.
- The phage-infected S. mutans emit quorum-sensing signal will pass the message down to the E.coli terminus then trigger the S. mutans killing module of it.
- luxR-luxI system embedded into S. mutans and E. coli respectively constructs the communicative links between the two different germs.
- The part reaches effective control on the number of S. mutans in case of that the threshold point defined by S. mutans’ number is surpassed.
purpose
background
design
Functional Measurement
result
Purpose
Targeting at the tooth problems, we are absolutely not going to rely on only one shoot on it. Our team is seeking for combinations of remedies to reach certain synergistic effect on preventing oral cavity.
Background
The two main organisms we adapted in our project are E. coli and M102 bacteriophage, respectively. Our team comes up with an interesting thought that we want to let these two entirely different organisms collaborate, thus we decided to use one of the most commonly seen quorum sensing chemicals, AHL, N-Acyl-homoserine-lactone, as our indicator between phage and E.coli. Due to the idea of phage threshold control of the number of S. mutans in prevention of the second-dominant strain in the oral cavity, we envision a possibility to trigger another killing system to reach synergistic effect on diminishing S. mutans. If the threshold terminators were passed, the downstream signaling pathway would be switched on and contact with the engineered E.coli.
Design
The circuit design of “communication module” could be divided into two parts, one in Streptococcus mutans, and the other in Escherichia coli.
The signal producer: Streptococcus mutans.
LuxI(BBa_C0061):
Coding sequence lies after the threshold terminator of nlmc promoter. LuxI acts as a gene which would generate AHL-synthase that yielding the quorum-sensing signal AHL right after the threshold level of S.mutans is exceeded.
Terminator(BBa_B0015):
Double terminator combined by terminator BBa_B0010 and BBa_B0012.
Signal receptor and killer module: Escherichia coli.
Constitutive promoter + RBS:
Sequences acquired by primer PCR from the template BBa_K523013 acts as promoter and ribosome binging site.
LuxR(BBa_C0062):
Coding sequence for LuxR protein that would complex with the quorum-sensing signal AHL. and then turns into an activated form which will afterwards bind with the promoter pLuxR and triggers it.
Terminator(BBa_B0015):
Double terminator combined by terminator BBa_B0010 and BBa_B0012.
pLuxR promoter(BBa_R0062):
Coding sequence for an inducible promoter pLuxR that would open the downstream killer module of Escherichia coli targeting at the cell walls of Streptococcus mutans in order to cause cell death of it.
Functional Measurement
To make a proof of our circuit design, we construct the functional test as a four-stages program.
Stage one: Circuit construction check.
After all the fragments of the circuit are linked, we need to test whether all the fragments are well-connected. Thus we design to insert red fluorescent protein gene (BBa_J06602) at the end of our coding sequence to ensure our whole circuit is constructed correctly.
Stage two: AHL releasing test.
Our main concern occurs that AHL is a gram-negative quorum sensing-signal yet we are going to let the engineered S.mutans which is actual a gram-positive bacteria to express and transmit AHL. In this stage, we are going to make a standard curve of the concentration of the commercialized AHL versus the absorption value first. Then transform the circuit containing LuxI into Streptococcus mutans. By extracting the supernatant of the culturing medium of the engineered S.mutans, and use spectrometer to measure the concentration of AHL compares to the standard curve made previously.
Stage three: Killer module inducing test.
In this stage, we are adding different amount of commercialized AHL to the engineered Escherichia coli implanted with our constructed circuit. We need to co-culture the E.coli and the normal Streptococcus mutans without our constructed circuit in this functional test program. The anticipating result will be reflected by the decrease of the number of normal S.mutans if the killer module is successfully activated by the quorum-sensing signal AHL.
Stage four: Final test with all combinations of the constructed circuits.
In this last stage we would put all the constructed circuits respectively into Escherichia coli and Streptococcus mutans. Co-culture of E.coli and S.mutans is also required in this testing program to make sure our expecting interaction between the two different engineered germs is conducted and well-functioned.
Result
Reference
