Part:BBa_K2607001:Experience

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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
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