Difference between revisions of "Team:UI Indonesia/Project"

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<h3><b>Methods</b></h3>
 
<h3><b>Methods</b></h3>
 
<h5>Our lab team started to use traditional cloning for insertion of desired gene into backbone plasmids for expression and iGEM 2018 submission. The traditional cloning would involve the methods of inserting gene fragments into MCS (multiple site cloning) of backbone or between prefix-suffix. The whole processes of gene cloning for further assays in this project are described in the <b>Figure 1 and 2</b>.
 
<h5>Our lab team started to use traditional cloning for insertion of desired gene into backbone plasmids for expression and iGEM 2018 submission. The traditional cloning would involve the methods of inserting gene fragments into MCS (multiple site cloning) of backbone or between prefix-suffix. The whole processes of gene cloning for further assays in this project are described in the <b>Figure 1 and 2</b>.
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<h5><b>Figure 1.</b> Principle methods of traditional cloning</h5>
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Revision as of 09:30, 16 October 2018

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
Our Protocols can be accessed here
Our Lab Notes can be accessed here
To be added
RESULTS AND DISCUSSIONS

Introduction


Managing the realization of the usage of synthetic diphtheria exotoxin (i.e. DiphTox/DT) and chimeric HB-EGF/Tar (i.e. HT) receptor, our team proposed a focus on characterizing the part of each BioBrick’s function. This year, our team, would be more focusing on testing the binding ability of both BioBricks in living E. coli spheroplast. FRET system would proceed in the later project of Finding Diphthy next in iGEM 2019. Before heading on to the lab results, it is recommended to fully understand the structure of DT and HT gBlocks and their respective protein functions in Parts (https://2018.igem.org/Team:UI_Indonesia/Parts) and Model domain (https://2018.igem.org/Team:UI_Indonesia/Model) of UI_Indonesia Wiki page.

Materials and Equipment


Some of the materials are provided by IHVCB lab, as well as Promega sponsored kit and IDT.

Materials from 2018 DNA Distribution Kit
  • Part BBa_J04450 containing mRFP BioBrick in the pSB1C3 backbone

Materials provided by IHVCB lab UI
  • ddH2O or nuclease-free water (NFW)
  • Chemical substances, such as MgCl2, NaCl, CaCl2, etc
  • 96-well plate
  • 96-well plate reader (GloMaxTM– Multi Detection System)
  • Luria Bertani (LB) liquid or agar media
  • Competent cell of Escherichia coli strain K12 derivates, such as TOP10 and DH5α, also strain B derivate such as BL21(DE3).
  • Antibiotics consisting of chloramphenicol (Cam) and ampicillin (Amp)
  • Polymerase chain reaction (PCR) thermal cycler
  • Micropipettes and tips
  • Microtubes and centrifuge tubes
  • Ice and buckets
  • Electrophoretic horizontal (DNA) and vertical (protein) chamber, power supply
  • Vortex
  • Minicentrifuge and ultracentrifuge
  • Gel electrophoresis reader (Gel DocTM XR+ Gel Documentation System) and sodium dodecyl sulphate - polyacrilamide gel electrophoresis (SDS-PAGE) reader (ImageQuantTM)
  • Buffers and enzymes (restriction enzymes such as EcoRI and PstI, ligase, etc.)
  • Static incubator and roto-shaker
  • Heatblock machine
  • Autoclave and microwave machine

Methods

Our lab team started to use traditional cloning for insertion of desired gene into backbone plasmids for expression and iGEM 2018 submission. The traditional cloning would involve the methods of inserting gene fragments into MCS (multiple site cloning) of backbone or between prefix-suffix. The whole processes of gene cloning for further assays in this project are described in the Figure 1 and 2.
Figure 1. Principle methods of traditional cloning
To be added
Team UI Indonesia
  igemui2018@gmail.com