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

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 (GloMax^TM– 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 Doc^TM XR+ Gel Documentation System) and sodium dodecyl sulphate - polyacrilamide gel electrophoresis (SDS-PAGE) reader (ImageQuant^TM)
• 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

Figure 2.. Original plan of how our lab team conducted the whole experiment in establishing parts for iGEM submission and characterizing our parts: DiphTox or DT (BBa_K2607000) and HB-EGF/Tar chimeric receptor or HT (BBa_K2607001).

Part I: DT and HT Cloning

Upon receiving our parts (in gBlocks) from Integrated DNA Technologies, Inc. (IDT), we performed PCR to amplify the gBlocks. PCR cloning for all gBlocks used the hand-made designed forward (Fwd) and reverse (Rev) cloning primers (i.e. specifications could be seen in Parts page). Furthermore, cycling formula for PCR cloning and confirmation could be accessed in the lab notes, as we applied GoTaq^TM Long PCR enzyme as the Hi-Fi polymerase. The amplified gBlocks were then used as inserts to plasmid vectors. For HT BioBrick, IDT was unable to yield the full sequence in high purity, so we had to split HT into two fragments (HT-1 and HT-2), which would later be amplified, restricted with SalI, and ligated to obtain complete HT fragment

On the other hand, we also prepared vectors for carrying our parts. Backbone pSB1C3-mRFP (BBa_J04450) has been used widely in our process of traditional cloning, for it provides much sensitive selection upon transformed recombinant plasmids. Since this plasmid does not contain any available expression promoter for the designed BioBrick, our supervisor suggested the usage of pQE80L expression vector belonged to IHVCB lab for functional assays and analyses. Therefore, we used pSB1C3 as cloning vector for submission to iGEM Headquarters and pQE80L as cloning vector for expression

We conducted traditional cloning (restriction-ligation) method to introduce our previously amplified inserts into prepared vectors. Restriction digestion was done sequentially with EcoRI and PstI in total of 8 hours by using the same buffer (i.e. EcoRI buffer and bovine serum antigen (BSA) 1X) with a minimum DNA template of 10 µg. Desalting and low-melting agarose (LMA) 1% electrophoresis purification was done to further remove any possible contaminating enzymes and undesired polynucleotides. Ligation of both vectors and inserts were conducted by adding T4 ligase and its respective buffers to be later incubated 16⁰C overnight

Transformation of resultant recombinant plasmids was done in wild-type E. coli K-12 (for submission purpose) and BL21(DE3) (for characterization and validation purpose) via “transformation protocol” methods in the other page. To enhance selection of recombinant E. coli, the transformed products were spread into selective LB agar containing appropriate antibiotic. Antibiotic formulation was complied to the lab’s proven antibiotics sensitivity test. We solubilized the powdered chloramphenicol in ethanol 95% and ampicillin in distilled water until final concentration of 25 mg/ml and 100 mg/ml, respectively. They were then added into LB media with ratio of 1:1000. After spread into LB agar, the transformed products were then incubated at 370C overnight.

In the case of transformation with pSB1C3, to select the colony with desired inserts, we performed red-white screening. If the grown colonies were red, it indicated that the colonies were transformed by native pSB1C3-mRFP and we excluded the colonies. We only picked white colonies (indicated that mRFP had been successfully removed from pSB1C3 and possibly replaced by insert) to be further confirmed for desired insert presence by colony PCR. We used VF2 and VR primers (i.e. iGEM standard primers) for confirmation of inserts in pSB1C3, while we used our hand-made designed primers for confirmation of inserts in pQE80L.

Finally, we performed mini-prep plasmid isolation for any confirmed colonies with desired inserts in pSB1C3. We grew the colonies in LB liquid medium at 37⁰C shaken overnight. Sequencing was performed to confirm the sequence of inserts before submitted to iGEM Headquarters.

Part II: SDS-PAGE Confirmation of Expressing BioBricks

Confirmation of any expressing DT and HT protein in recombinant E. coli BL21(DE3) was done via SDS-PAGE after isopropyl-D-1-thiogalactopyranoside (IPTG) induction for 4 hours in 370C in terrific broth (TB) medium with ampicillin. Subsequent lysis of E. coli to expose the desired proteins was done chemically via ionic and temperature induction. For DT containing His-Tag at the C-terminus of the protein, we managed to do His-Tag purification using magnetic beads. Binding of the DT protein into the beads would be enhanced by adding NaCl 500 mM. Incubation and washing were done 3X to remove any protein debris. Elution of the beads would generate the purified DT protein to be analyzed in the SDS-PAGE.

Part III: DT-HT Binding Assay

Prior to this step, our team expressed HT in transformed E. coli BL21(DE3) with pQE80L-HT by IPTG induction. In addition, we also had to remove outer membrane of the E. coli. The membrane removal would enable the HT receptor (in inner membrane) exposed directly towards extracellular environment, and possibly detecting DT.

Binding assays of DT and HT was conducted within 96-well plates by measuring the absorbance of 600 nm. This absorbance index indicates amounts of E. coli spheroplasts that successfully bound into DT in various environmental conditions (i.e. pH, temperatures, and DT concentration variables). Incubation was done within 60 minutes and purified magnetically using the available His-Tag. The amount of HT receptor binds to DT correlates positively with the amount of spheroplasts available in the eluents. Therefore, OD₆₀₀ is used as the primary quantification of spheroplasts amounts in the eluents. Specific details regarding methods of binding assays could be accessed via protocol page

Part IV: Luminescence (ADP-Glo^TM Kinase) Assays

To prepare the samples, we incubated E. coli BL21(DE3) transformed with empty pQE80L and pQE80L-HT cultures in LB liquid medium with ampicillin (1000:1) overnight. We created four replicates for each culture. On the following day, 1 mL from each replicate (eight in total) was aliquoted into 4 mL fresh TB medium with ampicillin (1000:1) and 4 µL IPTG 1 M. The replicates were incubated at 37^oC, 220 rpm for four hours. Their OD₆₀₀ were then determined and the replicates were subsequently pelleted at 12,000 rpm for one minute. The pellets were then lysed using Promega FastBreak^TM cell lysis reagent according the manufacturer protocol.

The following luminescence assay procedures is based on kit protocol with some modifications. First, we created standard curve according to the kit protocol to estimate ATP-to-ADP conversion rate from luminescence data. This performed by creating series of 1 mM ATP+ADP mixture with varying percentage of ADP relative to ATP+ADP (Table 1) in first row of microplate well. Series of 100 µM, 10 µM, and 1 µM were created by serial dilution in subsequent rows. The luminescence was then determined using Promega GloMax®-Multi Detection System.

Table 1.ATP+ADP mixture with varying percentage of ADP relative to ATP+ADP in microplate wells for standard curve.

Next, the reaction between lysed cells and purified DT with the amount of 180 nM (i.e. from Part III we found that Kd for HB-EGF/Tar and DT binding is 88.43 nM in 250C, then the maximum binding activity in 250C is expected to be achieved at twice of Kd) was carried out under 1x kinase reaction buffer consisting of 40 mM Tris pH 7.5, 20 mM MgCl2, and 0.1 mg/mL BSA. Ten µL from each reaction was aliquoted into wells in microplate and 10 µL of ADP-GloTM reagent was added to each well to stop the reaction and deplete the remaining ATP in the samples. After 40 minutes of incubation, 20 µL of kinase detection reagent was added to each well to convert ADP to ATP which will be used to generate luminescence and introduce substances (i.e. luciferin and luciferase) for luminescence. After 30 minutes of incubation, the luminescence was measured. Further details regarding ADP-Glo methods could be accessed in the protocol page.

To be added

Team UI Indonesia
  igemui2018@gmail.com