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
Genetic circuit
The gene for the parts used in our project are all expressed through a constitutive promoter and the inducible Lac promoter. The product of the genes do not interact with and regulate the expression of the gene of another part itself. Product of genes only interact with products of other genes.
The system made use of the chemotactic pathway in E. coli used to detect the presence of amino acids. The first part of the original pathway is the Tar receptor which detected aspartate. In our project, the receptor domain of this protein is modified by inserting the receptor domain of HB-EGF, the receptor which diphtheria toxin binds in human cells. The design of the chimeric protein, which we named HBEGF-Tar, is explained more deeply in the modelling page.
In the native pathway, when no aspartate binds the Tar receptor, the CheA protein autophosphorylates and interacted with the CheY protein. CheY then interacts with FliM protein in the motor section of the flagella. This interaction results in the tumbling movement of the flagella. When aspartate binds with the Tar receptor, the autophosphorylation of CheA stops, thus stopping the interaction of CheA with CheY. The interaction between CheA and CheY is what our team use to detect the binding of diphtheria toxin through the Lux-BRET (Bioluminescence Resonance Energy Transfer) system. For this purpose, we linked CheA to LuxAB protein and CheY to enhanced yellow fluorescence protein (eYFP).
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
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 GoTaqTM 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-GloTM 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 37oC, 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 FastBreakTM 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.
Result and Discussion
Part I: Gel Electrophoresis Confirmation of DT and HT cloning
Confirmation Insertion of DT into pQE80L and pSB1C3
Insertion of DT into pQE80L (for assays) and pSB1C3 (for submission) was done via traditional cloning using EcoRI and PstI restriction endonucleases. We have managed to maintain the workflow as preceding flowchart mentioned in methods. Transformation of recombinant plasmids is done into E. coli strain BL21(DE3) for pQE80L and TOP10 for pSB1C3, respectively. BL21(DE3) was chosen rather than TOP10 for assays, as it maintains anti-protease activity which would enable DT proteins undegraded intracellularly. In addition, BL21(DE3) is also a strain compatible for expression under T7 promoter (which is present in HT gBlock). Electrophoresis of colony PCR in the chamber utilized agarose 1% m/v soaked with TAE 1X, powered within 100 V for 30 minutes. Figure 3 and 4 shows the process of how we selected the colonies with recombinant plasmids, while Figure 5 and 6 were the final results of DT colony PCR confirmation.
Figure 3. Colonies of E. coli BL21(DE3) with pQE80L-DT in LB agar containing ampicillin
Figure 4. Ultraviolet (UV) illumination of E. coli transformed with pSB1C3-DT. Red-white screening could be utilized since our team used pSB1C3-mRFP as initial backbone vector.
Figure 5. Gel analysis of colony PCR on pQE80L-DT transformed into E. coli BL21(DE3). PCR colony primer would amplify ~250 bp bands that located inside the DT BioBrick, indicating that the DT fragment was successfully cloned into pQE80L.
Figure 6. Gel analysis of PCR colonies on pSB1C3-DT transformed E. coli TOP10. Universal flanked primers of VF2 and VR in pSB1C3 would amplify ~600 bp bands that were found in several following colonies, indicating that the DT fragment was successfully cloned into pSB1C3.
From these results, we concluded that DT BioBrick was successfully inserted into pSB1C3 and pQE80L backbone.
To be added
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
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 GoTaqTM 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-GloTM 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 37oC, 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 FastBreakTM 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.
Result and Discussion
Part I: Gel Electrophoresis Confirmation of DT and HT cloning
Confirmation Insertion of DT into pQE80L and pSB1C3
Insertion of DT into pQE80L (for assays) and pSB1C3 (for submission) was done via traditional cloning using EcoRI and PstI restriction endonucleases. We have managed to maintain the workflow as preceding flowchart mentioned in methods. Transformation of recombinant plasmids is done into E. coli strain BL21(DE3) for pQE80L and TOP10 for pSB1C3, respectively. BL21(DE3) was chosen rather than TOP10 for assays, as it maintains anti-protease activity which would enable DT proteins undegraded intracellularly. In addition, BL21(DE3) is also a strain compatible for expression under T7 promoter (which is present in HT gBlock). Electrophoresis of colony PCR in the chamber utilized agarose 1% m/v soaked with TAE 1X, powered within 100 V for 30 minutes. Figure 3 and 4 shows the process of how we selected the colonies with recombinant plasmids, while Figure 5 and 6 were the final results of DT colony PCR confirmation.
Figure 3. Colonies of E. coli BL21(DE3) with pQE80L-DT in LB agar containing ampicillin
Figure 4. Ultraviolet (UV) illumination of E. coli transformed with pSB1C3-DT. Red-white screening could be utilized since our team used pSB1C3-mRFP as initial backbone vector.
Figure 5. Gel analysis of colony PCR on pQE80L-DT transformed into E. coli BL21(DE3). PCR colony primer would amplify ~250 bp bands that located inside the DT BioBrick, indicating that the DT fragment was successfully cloned into pQE80L.
Figure 6. Gel analysis of PCR colonies on pSB1C3-DT transformed E. coli TOP10. Universal flanked primers of VF2 and VR in pSB1C3 would amplify ~600 bp bands that were found in several following colonies, indicating that the DT fragment was successfully cloned into pSB1C3.
From these results, we concluded that DT BioBrick was successfully inserted into pSB1C3 and pQE80L backbone.
To be added
- 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
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 GoTaqTM 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-GloTM 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 37oC, 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 FastBreakTM 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.
Result and Discussion
Part I: Gel Electrophoresis Confirmation of DT and HT cloning
Confirmation Insertion of DT into pQE80L and pSB1C3
Insertion of DT into pQE80L (for assays) and pSB1C3 (for submission) was done via traditional cloning using EcoRI and PstI restriction endonucleases. We have managed to maintain the workflow as preceding flowchart mentioned in methods. Transformation of recombinant plasmids is done into E. coli strain BL21(DE3) for pQE80L and TOP10 for pSB1C3, respectively. BL21(DE3) was chosen rather than TOP10 for assays, as it maintains anti-protease activity which would enable DT proteins undegraded intracellularly. In addition, BL21(DE3) is also a strain compatible for expression under T7 promoter (which is present in HT gBlock). Electrophoresis of colony PCR in the chamber utilized agarose 1% m/v soaked with TAE 1X, powered within 100 V for 30 minutes. Figure 3 and 4 shows the process of how we selected the colonies with recombinant plasmids, while Figure 5 and 6 were the final results of DT colony PCR confirmation.
Figure 3. Colonies of E. coli BL21(DE3) with pQE80L-DT in LB agar containing ampicillin
Figure 4. Ultraviolet (UV) illumination of E. coli transformed with pSB1C3-DT. Red-white screening could be utilized since our team used pSB1C3-mRFP as initial backbone vector.
Figure 5. Gel analysis of colony PCR on pQE80L-DT transformed into E. coli BL21(DE3). PCR colony primer would amplify ~250 bp bands that located inside the DT BioBrick, indicating that the DT fragment was successfully cloned into pQE80L.