Protein_Domain

Part:BBa_K2607000:Experience

Designed by: Andrea Laurentius   Group: iGEM18_UI_Indonesia   (2018-09-25)
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[iGEM 2018 UI_Indonesia] Finding Diphthy: Experiment with HB-EGF/Tar (HT) Receptor (BBa_K2607001)

We performed experiment to study the interaction between DiphTox (DT) (BBa_K2607000) and HB-EGF/Tar (HT) receptor (BBa_K2607001) using binding assay and luminescence (Promega's ADP-GloTM 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. Colonies of E. coli BL21(DE3) with pQE80L-DT in LB agar containing ampicillin.
Figure 3. 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 4. 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 5. 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.

Figure 2 and 3 shows the process on how we selected the colonies with possible recombinant plasmids, while Figure 4 and 5 shows the final results of DT colony PCR confirmation. From these results, we concluded that DT BioBrick was successfully inserted into pSB1C3 and pQE80L backbone.

Figure 6. Gradient temperature PCR for both HT-1 and HT-2 fragments. Note that more than 2 bands could be found in HT-1 and HT-2 fragment. 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 were conducted by using SalI restriction enzyme, yielding approximately 1954 bp complete HT BioBrick. Unfortunately, PCR amplification 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 6). Therefore, the gBlocks were shipped to Nanyang Technology University, Singapore (NTU-Singapore) team for ligation into pcDNA3 and pSB1C3. Our team conducted immediate transfer of complete HT BioBricks into pQE80L, while waiting for NTU finishing the cloning of the HT complete fragments into pSB1C3.

Figure 7. Colonies of E. coli BL21(DE3) transformed with pQE80L-HT in LB agar medium with ampicillin (1000:1).
Figure 8. Gel analysis of colony PCR on pQE80L-HT transformed E. coli BL21(DE3). The subsequent ~600s bp bands were found in the following colonies, indicating that the HT fragment was successfully inserted.
Figure 9. 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 7 and 8, while the transfer of completed HT into pSB1C3 (done by NTU-Singapore) is shown in Figure 9. These results suggested that HT BioBrick was successfully cloned into pSB1C3 and pQE80L backbone.

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 10. SDS-PAGE analysis (photographed via ImageQuantTM) of pQE80L-DT expression in E. coli BL21(DE3). White arrow indicates LacZα (size ~20.7 kDa) protein expression due to IPTG induction, indicating our IPTG was in good condition. On the other hand, black arrow shows DT (size ~7 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-DT(0) = E. coli BL21(DE3) containing recombinant pQE80L-DT with no IPTG induction; pQE80L-DT(0)p = purified protein of E. coli BL21(DE3) containing recombinant pQE80L-DT with no IPTG induction; pQE80L-DT(4) = E. coli BL21(DE3) containing recombinant pQE80L-DT with 4 hours of IPTG induction; pQE80L-DT(4)p = purified protein of E. coli BL21(DE3) containing recombinant pQE80L-DT after 4 hours of IPTG induction.
Figure 11. SDS-PAGE analysis (photographed via ImageQuantTM) 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.

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