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[iGEM 2018 UI_Indonesia] Finding Diphthy: Experiment with DiphTox (DT) (BBa_K2607000)

We performed experiment to study the interaction of HB-EGF/Tar (HT) receptor (BBa_K2607001) with DiphTox (DT) (BBa_K2607000) using binding assay and luminescence (Promega's ADP-Glo^TM Kinase) assay. Figure 1 shows complete workflow of this experiment.

Figure 1. Original plan workflow to study interaction between DT (BBa_K2607000) and HT (BBa_K2607001).

DT and HT Cloning
Upon receiving DT and HT (in gBlocks) from Integrated DNA Technologies, Inc. (IDT), we performed PCR to amplify the gBlocks. PCR amplification for all gBlocks used the designated forward (Fwd) and reverse (Rev) cloning primers. Furthermore, cycling formula for PCR cloning and confirmation could be accessed in the iGEM 2018 UI_Indonesia team's lab notes[http://2018.igem.org/Team:UI_Indonesia/Notebook], 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 Institute of Human Virology and Cancer Biology (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 Escherichia coli K-12 (for submission purpose) and BL21(DE3) (for characterization and validation purpose). 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.

Figure 2. Gradient temperature PCR for both HT-1 and HT-2 fragments. Note that more than one bands could be found in lanes containing PCR amplification of HT-1 and HT-2 fragments. These amplified bands explain that the synthetically designed primers were not specific enough. Original HT-1 fragment is 1105 bp in size, while HT-2 fragment size is 864 bp.

While we had no difficulties with DT, the methods for inserting complete HT fragment were quite challenging, since it must be linearly ligated in the first place prior to recombination into plasmid vector. Linear ligation of HT-1 and HT-2 fragments cut with SalI restriction enzyme would yield approximately 1954 bp complete HT BioBrick. Unfortunately, PCR amplification of HT-1 and HT-2 fragments in the beginning using hand-made designed Fwd and Rev cloning primers could not generate specific bands (i.e. the primers anneals unspecific in various length of both HT fragments, Figure 2). Therefore, the gBlocks were shipped to Nanyang Technology University, Singapore (NTU-Singapore) team for ligation into pcDNA3 and pSB1C3[http://2018.igem.org/Team:UI_Indonesia/Collaborations]. Our team conducted immediate transfer of complete HT BioBricks from pcDNA3 into pQE80L, while waiting for NTU finishing the cloning of the HT complete fragments into pSB1C3.

Figure 3. Colonies of E. coli BL21(DE3) transformed with pQE80L-HT in LB agar medium with ampicillin (1000:1).

Figure 4. Gel analysis of colony PCR on pQE80L-HT transformed E. coli BL21(DE3) with Fwd and Rev Colony PCR Primer for HT-1 and HT-2 ligation confirmation. We did not use Fwd and Rev Colony PCR Primer for complete HT because we failed to yield expected 1836 bp bands (we suspected this was due to our PCR enzyme limitation). The subsequent ~600s bp bands were found in the following colonies, indicating that the HT-1 and HT-2 fragment were successfully ligated and complete HT was successfully inserted into pQE.

Figure 5. Gel analysis of colony PCR of pSB1C3-HT. Universal VF2 and VR primers would amplify ~2000s bp bands that located inside the BioBrick HT, indicating that the HT fragment was successfully cloned into pSB1C3. Note: this datum was provided by team NTU-Singapore as gratitude for their aid in submission cloning for team UI_Indonesia’s HT fragments.

The transfer of completed HT into pQE80L is shown in Figure 3 and 4, while the transfer of completed HT into pSB1C3 (done by NTU-Singapore) is shown in Figure 5. These results suggested that HT BioBrick was successfully cloned into pSB1C3 and pQE80L backbone. As for DT cloning, the results can be seen from DT Registry Page[1].

Sodium Dodecyl Sulphate-Polyacrilamide Gel Electrophoresis (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.

Figure 6. SDS-PAGE analysis (photographed via ImageQuant^TM) of pQE80L-HT expression in E. coli BL21(DE3). White arrow indicates LacZα (size ~20.7 kDa) protein expression due to IPTG induction, while black arrow shows increasing HT (size ~57.8 kDa) protein expression as it is induced with IPTG within 4 hours. Note: pKS(0) = E. coli TOP10 transformed with pBluescript KS(-) with no IPTG induction; pKS(4) = E. coli TOP10 transformed with pBluescript KS(-) after 4 hours of IPTG induction in 370C; BL21(DE3) = wild-type E. coli BL21(DE3); BL21(DE3) w/ pQE80L = E. coli BL21(DE3) containing empty pQE80L; pQE80L-HT(0) = E. coli BL21(DE3) containing recombinant pQE80L-HT with no IPTG induction; pQE80L-HT(4) = E. coli BL21(DE3) containing recombinant pQE80L-HT after 4 hours of IPTG induction.

After insertion of both BioBricks into pQE80L, the assays could begin with expression confirmation. For transcription initiation of BioBricks require lac promoter provided by the vector, induction of IPTG was essential. Identification of positive control using E. coli TOP10 transformed with pBluescript KS(-) could be important in determining whether our IPTG used was expired or not. Wild-type E. coli BL21(DE3) and E. coli BL21(DE3) inserted with empty pQE80L were used as negative control. Furthermore, purification of DT was conducted to increase sensitivity of expression yield. Figure 6 shows the SDS-PAGE performed to confirm HT expression, while DT expression confirmation can be seen in DT Registry Page[2]. From these results, we concluded that we successfully expressed DT and HT.

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.

Upon confirmation of DT and HT expression, our team would like to testify the interaction of those proteins. Prior to binding assay, the recombinant E. coli possessing HT expression should be uncoated from the outer membrane layer. This lets huge molecules or proteins accessing the periplasmic layer or inner plasma membrane of the bacteria. Transforming E. coli into intact spheroplast could be a disadvantageous for the cell itself, since the membrane is more fragile to extracellular extremes. This would be one of the major challenges of the binding assays in determining specific pH and temperatures for keeping the spheroplasts alive. Methods for making spheroplasts could be accessed in the protocol page. Spheroplasts were subjected to different DT concentration during one hour incubation with different temperatures (Table 1). Binding of HT receptor towards intact DT in Magne-His beads would prevent spheroplasts elimination during washing process. Elution of spheroplasts would be the final variable in quantifying DT-HT binding strength.

Table 1. Net OD₆₀₀ (minus blank: elution buffer) results of DT-HT binding assays in different temperatures and various DT concentrations. Triplicates were done to minimize bias of absorbance data.

DT Concentration (nM)	OD₆₀₀
	4⁰C					25⁰C
	Rep1	Rep2	Rep3	Mean	St. Dev.	Rep1	Rep2	Rep3	Mean	St. Dev.
100	0.07595	0.0765	0.075421	0.075957	0.00054	0.065845	0.066	0.066149	0.065998	0.000152
200	0.09625	0.0942	0.091866	0.094105	0.002194	0.091028	0.0904	0.089112	0.09018	0.000977
300	0.116465	0.119	0.117009	0.117491	0.001335	0.11038	0.112	0.096269	0.106216	0.008653
400	0.134605	0.135	0.129117	0.132907	0.003288	0.119349	0.1258	0.124289	0.123146	0.003374
500	0.142971	0.142	0.13692	0.14063	0.00325	0.092539	0.1339	0.132041	0.119493	0.023362
600	0.131838	0.1308	0.136578	0.133072	0.00308	0.105433	0.108	0.109468	0.107634	0.002042
700	0.140814	0.1398	0.130853	0.137156	0.005482	0.120124	0.1125	0.109777	0.114134	0.005363
800	0.139218	0.1389	0.138472	0.138863	0.000374	0.112397	0.1146	0.104352	0.11045	0.005394
900	0.128478	0.1296	0.124337	0.127472	0.002772	0.113375	0.1076	0.106478	0.109151	0.003701
1000	0.136597	0.1334	0.12997	0.133322	0.003314	0.110781	0.1035	0.102042	0.105441	0.004682
DT Concentration (nM)	OD₆₀₀
	37⁰C					50⁰C
	Rep1	Rep2	Rep3	Mean	St. Dev.	Rep1	Rep2	Rep3	Mean	St. Dev.
100	0.052625	0.0523	0.052395	0.05244	0.000167	0.032055	0.0403	0.0403	0.035964	0.004139
200	0.081469	0.0815	0.081521	0.081497	0.000026	0.058493	0.05687	0.05687	0.057857	0.000867
300	0.098739	0.0986	0.098208	0.098516	0.000275	0.053524	0.0535	0.0535	0.053563	0.000089
400	0.121782	0.1212	0.120972	0.121318	0.000418	0.055359	0.0561	0.0561	0.055714	0.000371
500	0.129521	0.1066	0.109124	0.115082	0.012568	0.055447	0.0665	0.0665	0.064436	0.008156
600	0.114763	0.1177	0.118085	0.116849	0.001817	0.065657	0.0682	0.0682	0.067563	0.00168
700	0.111707	0.1142	0.115469	0.113792	0.001914	0.069064	0.0703	0.0703	0.070139	0.001005
800	0.127036	0.1302	0.133242	0.130159	0.003103	0.068916	0.0706	0.0706	0.070327	0.001296
900	0.093508	0.1259	0.13026	0.116556	0.020079	0.073543	0.0749	0.0749	0.074389	0.000738
1000	0.119084	0.1204	0.118508	0.119331	0.00097	0.076607	0.0767	0.0767	0.076858	0.000357

Figure 7. Average amount of E. coli spheroplasts (expressed in OD₆₀₀) remained in the eluent after magnetic binding with DT in different DT concentrations and temperatures. OD₆₀₀ is expressed within arbitrary unit (a.u.). Standard error of estimates is shown in between 0.005-0.008.

Figure 8. P-P plot regression analysis to determine its Lineweaver-Burke linear equation. R square values and linear equations are shown within regression in each temperature via Statistical Package for Social Sciences (SPSS) Basic Statistics v.21. Therefore, standardized values of K_d and OD₆₀₀ max could be determined in different temperature conditions.

Plotting of the OD₆₀₀ in the graph (Figure 7) could be referred classically as ligand-receptor dynamics. The increasing of DT concentration would enable huge amount binding of spheroplasts in the incubation. Therefore, the logarithmic trend occurs in the beginning. Nevertheless, there would be an amount of DT concentration where the number of spheroplast binding would be reaching its maximum. Affinity constant of the receptor towards ligands could be determined as the amount of substrate causing saturation of 50% receptors. Reverse plotting of Lineweaver-Burke would be required to determine the values of HT-DT affinity constant and maximum saturation. Modified receptor-ligand formulae could be further transformed as shown in Figure 8. (Note: y represents 1/OD₆₀₀, and x represents 1/substrate concentration in linear equation).

Different temperatures exhibit different affinity constant and maximum binding (Table 2). It is natural that HT receptor possesses optimum tertiary structure facilitating binding of DT in certain environmental condition. Highest affinity of HT towards DT occurs in temperature of 25⁰C. Additionally, maximum binding of HT towards DT happens highest in 4⁰C and 37⁰C. Low temperature might increase the possibility of HT-DT binding, since the molecular kinetical energy is low. Therefore, extrapolation of affinity and maximum binding from different temperatures into single human body temperatures (37⁰C) would be required in future research. In conclusion, positive binding of HT and DT shows that the designed ligand-receptor could be further used as predicted models of diphtheria toxins interaction in human body.

Table 2. Effect of temperatures towards binding kinetics of DT and HT.

Temperature (⁰C)	Affinity Constant K_d (nM)	Maximum Binding OD₆₀₀(a.u.)
4	106.694	0.1556
25	88.43	0.1285
37	187.39	0.1541
50	122.66	0.0813

Profound understanding of the natural HT and DT binding could be analyzed by measuring the binding activities in different pH concentration. This could serve as potential basis for prediction models of environment that supports the interaction. Principles of measuring the activities are the same as previous one. Triplicates are subjected to different pH solution, ranging from 4-8.5, during one hour incubation prior to elution (Table 3). The used concentration of DT in this assay was 180 nM (as it referred to maximum binding of DT concentration towards HT in 25⁰C).

Table 3. Net OD₆₀₀ (minus blank: elution buffer) results of DT-HT binding in different extracellular pH. Triplicates were conducted to minimize bias effect of absorbance index.

OD₆₀₀	pH
OD₆₀₀	4	4.5	5	5.5	6	6.5	7	7.5	8	8.5
Rep1	0.075619	0.08983	0.078769	0.05905	0.076684	0.088825	0.12357	0.10124	0.090671	0.080905
Rep2	0.073124	0.091508	0.080834	0.05912	0.07562	0.087584	0.126196	0.101931	0.088198	0.082509
Rep3	0.073049	0.09254	0.081135	0.059364	0.076381	0.084683	0.128242	0.102149	0.087184	0.084606
Mean	0.073931	0.091293	0.080246	0.059178	0.076228	0.087031	0.126003	0.101773	0.088684	0.082673
St. Dev.	0.001463	0.001368	0.001288	0.000165	0.000548	0.002126	0.002342	0.000475	0.001794	0.001856

Figure 9. This clustered column represents amount of spheroplasts remained in the eluent buffer after washing in different pH solution. Triplicates with standard bar errors indicate any data bias optimized as samples. Different alphabetic indexes top of the column express data significances, such as a-b (p<0.05), a-c (p<0.05), and b-c (p<0.01).

Optimal pH for the binding interaction occurs at 7 to 7.5 (Figure 9). This is exactly the physiological pH condition inside human body. Significant drop of binding activity observed at pH 5.5 could indicate early spheroplast autolysis, since it triggers intracellular cascade of lysis protein according to Raam R, et al. Therefore, lab models of binding HT-DT could be conducted optimally within pH range of 7 to 7.5.

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 37⁰C, 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 4) 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 4. ATP+ADP mixture with varying percentage of ADP relative to ATP+ADP in microplate wells for standard curve.

Reagent Added	Volume (µL)
Reagent Added	100%	80%	60%	40%	20%	10%	5%	4%	3%	2%	1%	0%
ADP 1 mM	100	80	60	40	20	10	5	4	3	2	1	0
ATP 1 mM	0	20	40	60	80	90	95	96	97	98	99	100

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 K_d) 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.

Figure 10. Standard curve for percent ATP-to-ADP conversion from luminescence with varying concentrations of ATP+ADP mixture (1 µM, 10 µM, 100 µM, and 1 mM). RFU = relative light units.

The standard curve obtained for percent ATP-to-ADP conversion from luminescence data is shown in Figure 10. With this standard curve, we can estimate how much ATP is converted into ADP (or how much ATP is conserved in HT-DT reaction compared to normal) based on given luminescence data later in the experiment.

Table 5. Net OD₆₀₀ (minus blank) and luminescence (in RFU) after reaction with DT.

Sample		Net OD₆₀₀	Luminescence (RFU)	Luminescence per OD₆₀₀(RFU)
Control (E. coli BL21(DE3) + empty pQE80L)	Replicate 1	0.297	557028	1875515
	Replicate 2	0.285	637420	2236561
	Replicate 3	0.274	594330	2169088
	Replicate 4	0.292	615446	2107692
	Mean			2097214
	Standard Deviation			156890.3
Experimental (E. coli BL21(DE3) + pQE80L + HT)	Replicate 1	0.289	427903	1480633
	Replicate 2	0.301	492191	1635186
	Replicate 3	0.308	459287	1491192
	Replicate 4	0.293	470488	1605761
	Mean			1553193
	Standard Deviation			78730.27

Figure 11. Luminescence per OD₆₀₀ (RFU) of control (transformed E. coli BL21(DE3) with empty pQE80L) and experimental (transformed E. coli BL21(DE3) with pQE80L+HT) group. * indicates significant difference compared to control (p = 0.001).

Table 5 shows net OD₆₀₀ (after deducted by blank Abs600) and their luminescence after reaction with DT for each replicate, while Figure 11 shows comparison between luminescence per OD₆₀₀ for control (E. coli BL21(DE3) transformed with empty pQE80L) and experimental (E. coli BL21(DE3) transformed with pQE80L+ HT) group upon reaction with DT. We found that luminescence level in experimental group was significantly lower than control group (p = 0.001). This result suggested that experimental group has lower ADP concentration than control group, which likely to be caused by HT interaction with DT inhibits CheA phosphorylation, ultimately leading to more ATP conserved and less ADP produced. Therefore, upon reaction with kinase detection reagent, less ADP will be converted into ATP in experimental group, causing less luminescence compared with control group.

From the experiment, we did not know the initial ATP+ADP concentration from each control and experimental group. Assuming the initial ATP concentration was 1 mM (mean ATP concentration in E. coli is 1.56 ± 1.27 mM according to Yaginuma et al.), with the help of standard curve from Figure 1, we can estimate that percent of ATP-to-ADP conversion inhibition upon HT-DT interaction was around 1%. This is due to luminescence of standard 0% ADP-ATP mixture was 1.69 x 10⁶, similar with mean luminescence of experimental group (1.55 x 10⁶), while luminescence of standard 1% ADP-ATP mixture was 2.2 x 10⁶, similar with mean luminescence of control group (2.1 x 10⁶). One note to be considered that E. coli cells may have varying level of intracellular ATP and ADP, as stated by large standard deviation of ATP concentration from Yaginuma et al. study. However, we grew and treated E. coli used in this experiment equally, also we created replicates for each group to minimize the possible bias.

Hence, our present result suggested that our HT works as expected to inhibit phosphorylation, shown by less luminescence generated compared with control, indicating that less ADP was produced upon HT-DT interaction.

References

[3]Mohan, R. R., Kronish, D. P., Pianotti, R. S., Epstein, R. L., & Schwartz, B. S. (1965). Autolytic Mechanism for Spheroplast Formation in Bacillus cereus and Escherichia coli. Journal of Bacteriology, 90(5), 1355–1364.
[4]Yaginuma H, et al. Diversity of ATP concentrations in a single bacterial cell population revealed by quantitative single-cell imaging. 2014.

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