Team:UI Indonesia/Project

OVERVIEW

Diphtheria is becoming a prominent issue in Indonesia as its incidence is increasing recently. It also causes various complications, leading to morbidity and mortality. We realized the urgency of fast, reliable, and cheap early detection method for diphtheria infection as one of means necessary for its eradication. Therefore, we created a chimeric between native Escherichia coli Tar chemotaxis receptor and human HB-EGF receptor so the bacterium may recognize diphtheria toxin. Moreover, we combined CheA and CheY in E. coli chemotaxis system with LuxAB and eYFP, respectively. When in contact, LuxAB and eYFP create resonance energy transfer system in which LuxAB gives its emission to eYFP. Without diphtheria toxin, CheA will be in phosphorylated state, allowing interaction with CheY and energy transfer, resulting in yellow color. Toxin binding into chimeric receptor will inhibit CheA phosphorylation, hindering interaction with CheY and energy transfer, resulting in blue color (i.e. LuxAB native color).

Pathogenesis of Diphtheria: How Does Corynebacterium diphtheriae Cause the Disease?

Corynebacterium diphtheriae is a Gram-positive rod bacterium that causes diphtheria. It produces exotoxin with two fragments (AB toxin). Fragment B facilitates toxin internalization within host cell via endocytosis upon binding HB-EGF receptor,while fragment A catalyzes modification of ribosomal elongation factor-2; therefore, it suppresses protein synthesis

Tar-mediated Chemotaxis System in Escherichia coli

In E. coli, chemotaxis mediated by methyl-accepting chemotaxis proteins (MCPs) has been widely studied. MCPs are transmembrane chemoreceptors with periplasmic ligand binding domain and cytoplasmic signaling domain. To date, four different E. coli MCPs have been identified: Tar, Tsr, Trg and Tap chemoreceptors.

Tar chemoreceptor mediates E. coli movement towards aspartate and maltose (attractant molecules). It undergoes conformational change upon attractant molecule binding, leading to inhibition of CheA protein autophosphorylation and subsequent CheY activity. This causes the flagellum facilitates the bacterium to swin towards attractant substance via opposite motor protein FliM rotation. On the other hand, CheA autophosphorylation would cause prominent rotation of motor protein to make tumbling effect.

LuxAB-eYFP Fluorescence Resonance Energy Transfer (FRET) System

Basically, a molecule is excited to higher energy state when it absorbs a photon energy. This molecule relaxes back to ground state when the energy is emitted back to the environment or transferred into another molecule. FRET is a phenomenon in which non-radioactive energy is transferred from excited donor molecule to acceptor molecule via dipole-dipole interactions. Molecules involved in this phenomenon are called fluorophores as they emit fluorescence according to their respective emission spectrum after absorbing higher photon energy. LuxAB and eYFP are one of the most widely studied paired fluorophores. In this case, LuxAB is the donor fluorophore as it emits cyan colored light with relatively high energy (peak emission at 490 nm). eYFP serves as the acceptor fluorophore when in close contact with LuxAB, as it absorbs high energy from LuxAB that is overlapped with its own absorption spectrum and emits yellow colored light with lower energy (peak emission at 530 nm). To be utilized in macromolecules interaction studies, LuxAB and eYFP should be incorporated with the molecules of interest. When the molecules of interest are in contact, energy transfer between LuxAB and eYFP will happen and its efficiency can be measured with fluorescence-lifetime imaging microscopy method.

OUR PROJECT

Luria Bertani Liquid Medium Protocol

1. Make LB medium using this recipe
1. a. Tryptone 10 g/L
2. b. NaCl 10 g/L
3. c. Yeast Extract 5 g/L
4. d. H2O untuk the volume reach 1 Liter
5. 2. Autoclave the solution

Luria Bertani Agar Protocol

1. Make LB Agar mix using this recipe

a. Agar powder 20 g/L
b. NaCl 10 g/L
c. Tryptone 10 g/L
d. Yeast Extract 5 g/L
e. H2O until the volume reach 1 Liter
1. 2. Autoclave LB agar
2. 3. Pour the solution into petri dish. each petri dish contain about 125 mL LB agar
   

   Polymerase Chain Reaction (PCR) Protocol

3. 1. Create PCR Master mix (total volume 50µL)
   a. 5µL Taq Reaction Buffer 10x
   b. 1µL 10mM dNTPs
   c. 1µL 10µM Forward primer
   d. 1µL 10µM Reverse Primer
   e. DNA Template
   f. 0.25µL Taq DNA Polymerase
   g. Nuclease Free Water until volume 50µL
   1. 2. Transfer PCR master mix tube into thermocycler
   2. 3. Set how many cycles the PCR will be
   3. 4. Thermocycler will begin initial denaturation steps with 95 oC for 30 seconds
   4. 5. Thermocycler will continue into these steps repeated based on how many cycles are set
      a. Denaturation : 95 oC for 15 – 30 seconds
      b. Annealing : 45 – 68oC for 15 – 60 seconds
      c. Elongation : 60 oC for 1 minute
      1. 6. Thermocycler will the proceed into final extension steps with 68 oC for 5 minutes
      2. 7. PCR are done

         To be added

         RESULTS AND DISCUSSIONS

         To be added

         Team UI Indonesia
           igemui2018@gmail.com