Team:AFCM-Egypt/Demonstrate
1. Parts Synthesis, Restriction and Ligation
We began building our construct by first synthesizing our parts and digesting them using IGEM plasmid pS1BC3 compatible restriction enzymes XbaI and PstI.as shown in figures 1-3.
Ligation protocol
XbaI Order R0145 PstI Order R0140 Restriction Enzyme Double Digestion Steps Set up reaction as follows: COMPONENT 50 µl REACTION DNA 1 µg 10X NEBuffer 3.1 5 µl (1X) XbaI 1.0 µl (or 10 units) PstI 1.0 µl (or 10 units) Nuclease-free Water to 50 µl Incubate at 37°C for 5-15 minutes as both enzymes are Time-Saver qualified.
Lane 1: DNA 100 bp Ladder MW marker
Lane 2a: Part BBa_K2534023 (CME E CMV P) gblock undigested fragment
Lane 2b: Part BBa_K2534023 (CME E CMV P) gblock digested fragment
Lane 3: Part BBa_K2534007 ( AMPp+AMPR )gblock digested fragment
Lane 4a: Part BBa_K2534011 (B glob intron + vsv G + poly a) gblock undigested fragment
Lane 4b: Part BBa_K2534011 (B glob intron + vsv G + poly a) gblock digested fragment
Lane 5a: Part BBa_K2534012 (HIV gag) gblock undigested fragment
Lane 5b: Part BBa_K2534012 (HIV gag) gblock digested fragment
Lane 6a: Part BBa_K2534013 (HIV pol) gblock undigested fragment
Lane 6b: Part BBa_K2534013 (HIV pol) gblock digested fragment
Lane 7a: Part BBa_K2534014 (RRE) gblock undigested fragment
Lane 7b: Part BBa_K2534014 (RRE) gblock digested fragment
Lane 8a: Part BBa_K2534015 (SV40p) gblock undigested fragment
Lane 8b: Part BBa_K2534015 (SV40p) gblock digested fragment
Lane 1: DNA 100 bp Ladder MW marker
Lane 9a: Part BBa_K2534017 (WRPE) gblock undigested fragment
Lane 9b: Part BBa_K2534017 (WRPE) gblock digested fragment
Lane 10a: Part BBa_K2534018 (KOZAK EGFP) gblock undigested fragment
Lane 10b: Part BBa_K2534018 (KOZAK EGFP) gblock digested fragment
Lane 11a: Part BBa_K2534022 (TetR) gblock undigested fragment
Lane 11b: Part BBa_K2534022 (TetR) gblock digested fragment
Lane 1: DNA 100 bp Ladder MW marker
Lane 2a: Part BBa_K2534016 (HIV LTR) gblock undigested fragment
Lane 2b: Part BBa_K2534016 (HIV LTR) gblock digested fragment
Lane 3a: Part BBa_K2534020 (CCP/PPt) gblock undigested fragment
Lane 3b: Part BBa_K2534020 (CCP/PPt) gblock digested fragment
Lane 4a: Part BBa_K2534019 (HV PSI) gblock undigested fragment
Lane 4b: Part BBa_K2534019 (HIV PSI) gblock digested fragment
Lane 5a: Part BBa_K2534021 (Tet op) gblock undigested fragment
Lane 5b: Part BBa_K2534021 (Tet op) gblock digested fragment
2.MiniPrep, Ligation and characterization of Parts ligated to IGEM Plasmid pS1BC3
We then performed our plasmid MiniPrep to ligate and characterize our parts to the plasmid pS1BC3 (figures 4-6).
MiniPrep protocol
The QIAprep Spin Miniprep Kit (cat. nos. 27104 and 27106) can be stored at room temperature (15–25°C) for up to 12 months. For more information, please refer to the most recent version of the QIAprep Miniprep Handbook, which can be found at: www.qiagen.com/handbooks. For technical assistance, please call toll-free 00800-22-44-6000, or find regional phone numbers at: www.qiagen.com/contact. Notes before starting - Optional: Add LyseBlue reagent to Buffer P1 at a ratio of 1 to 1000. - Add the provided RNase A solution to Buffer P1, mix and store at 2–8°C. - Add ethanol (96–100%) to Buffer PE before use (see bottle label for volume). - All centrifugation steps are carried out at 13,000 rpm (~17,900 x g) in a conventional table-top microcentrifuge. 1. Pellet 1–5 ml bacterial overnight culture by centrifugation at >8000 rpm (6800 x g) for 3 min at room temperature (15–25°C). 2. Resuspend pelleted bacterial cells in 250 μl Buffer P1 and transfer to a microcentrifuge tube. 3. Add 250 μl Buffer P2 and mix thoroughly by inverting the tube 4–6 times until the solution becomes clear. Do not allow the lysis reaction to proceed for more than 5 min. If using LyseBlue reagent, the solution will turn blue. 4. Add 350 μl Buffer N3 and mix immediately and thoroughly by inverting the tube 4–6 times. If using LyseBlue reagent, the solution will turn colorless. 5. Centrifuge for 10 min at 13,000 rpm (~17,900 x g) in a table-top microcentrifuge. 6. Apply 800 μl supernatant from step 5 to the QIAprep 2.0 spin column by pipetting. For centrifuge processing, follow the instructions marked with a triangle (). For vacuum manifold processing, follow the instructions marked with a circle (). Centrifuge for 30–60 s and discard the flow-through, or apply vacuum to the manifold to draw the solution through the QIAprep 2.0 spin column and switch off the vacuum source. 7. Recommended: Wash the QIAprep 2.0 spin column by adding 0.5 ml Buffer PB. Centrifuge for 30–60 s and discard the flow-through, or apply vacuum to the manifold to draw the solution through the QIAprep 2.0 spin column and switch off the vacuum source. Note: This step is only required when using endA+ strains or other bacteria strains with high nuclease activity or carbohydrate content. 8. Wash the QIAprep 2.0 spin column by adding 0.75 ml Buffer PE. Centrifuge for 30–60 s and discard the flow-through, or apply vacuum to the manifold to draw the solution through the QIAprep 2.0 spin column and switch off the vacuum source. Transfer the QIAprep 2.0 spin column to the collection tube. 9. Centrifuge for 1 min to remove residual wash buffer. 10.Place the QIAprep 2.0 column in a clean 1.5 ml microcentrifuge tube. To elute DNA, add 50 μl Buffer EB (10 mM TrisCl, pH 8.5) or water to the center of the QIAprep 2.0 spin column, let stand for 1 min, and centrifuge for 1 min. 11.If the extracted DNA is to be analyzed on a gel, add 1 volume of Loading Dye to 5 volumes of purified DNA. Mix the solution by pipetting up and down before loading the gel.
Ligation protocol
Set up the following reaction in a microcentrifuge tube on ice. (T4 DNA Ligase should be added last. Note that the table shows a ligation using a molar ratio of 1:3 vector to insert for the indicated DNA sizes.) Use NEBioCalculator to calculate molar ratios. COMPONENT 20 μl REACTION T4 DNA Ligase Buffer (10X)* 2 μl Vector DNA (4 kb) 50 ng (0.020 pmol) Insert DNA (1 kb) 37.5 ng (0.060 pmol) Nuclease-free water to 20 μl T4 DNA Ligase 1 μl* The T4 DNA Ligase Buffer should be thawed and resuspended at room temperature. Gently mix the reaction by pipetting up and down and microfuge briefly. For cohesive (sticky) ends, incubate at 16°C overnight or room temperature for 10 minutes. For blunt ends or single base overhangs, incubate at 16°C overnight or room temperature for 2 hours (alternatively, high concentration T4 DNA Ligase can be used in a 10 minute ligation). Heat inactivate at 65°C for 10 minutes. Chill on ice and transform 1-5 μl of the reaction into 50 μl competent cells.
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Lane 1: DNA 100 bp Ladder MW marker
Lane 2: Part BBa_K2534023 (CME E CMV P) gblock ligated to IGEM plasmid pS1BC3
Lane 3: Part BBa_K2534007 (AMPp+AMPR) gblock ligated to IGEM plasmid pS1BC3
Lane 4: Part BBa_K2534011 (B glob intron + vsv G + poly a) gblock ligated to IGEM plasmid pS1BC3
Lane 5: Part BBa_K2534012 (HIV gag) gblock ligated to IGEM plasmid pS1BC3
Lane 6: Part BBa_K2534018 (KOZAK EGFP) gblock ligated to IGEM plasmid pS1BC3
Lane 7: Part BBa_K2534022 (TetR) gblock ligated to IGEM plasmid pS1BC3
Lane 8: Part BBa_K2534014 (RRE) gblock ligated to IGEM plasmid pS1BC3
Lane 9: Part BBa_K2534011 (B glob intron + vsv G + poly a) gblock religated to IGEM plasmid pS1BC3
Lane 10: Part BBa_K2534011 (B glob intron + vsv G + poly a) gblock religated to IGEM plasmid pS1BC3
Lane 11: Part BBa_K2534012 (HIV gag) gblock re-ligated to IGEM plasmid pS1BC3
Lane 12: Part BBa_K2534012 (HIV gag) gblock re-ligated to IGEM plasmid pS1BC3
Lane 13: IGEM plasmid backbone pSB1C3
Lane 1: DNA 100 bp Ladder MW marker
Lane 2: Part BBa_K2534012 (HIV gag) gblock re-ligated to IGEM plasmid pS1BC3
Lane 3: Part BBa_K2534012 (HIV gag) gblock re-ligated to IGEM plasmid pS1BC3
Lane 4: Part BBa_K2534013 (HIV pol) gblock ligated to IGEM plasmid pS1BC3
Lane 5: Part BBa_K2534014 (RRE) gblock ligated to IGEM plasmid pS1BC3
Lane 6: Part BBa_K2534014 (RRE) gblock ligated to IGEM plasmid pS1BC3
Lane 7: Part BBa_K2534015 (SV40p) gblock ligated to IGEM plasmid pS1BC3
Lane 8: Part BBa_K2534016 (HIV LTR) gblock ligated to IGEM plasmid pS1BC3
Lane 9: Part BBa_K2534016 (HIV LTR) gblock re-ligated to IGEM plasmid pS1BC3
Lane 10: Part BBa_K2534017 (WRPE) gblock ligated to IGEM plasmid pS1BC3
Lane 11: Part BBa_K2534014 (RRE) gblock re-ligated to IGEM plasmid pS1BC3
Lane 12: Part BBa_K2534014 (RRE) gblock re-ligated to IGEM plasmid pS1BC3
Lane 13: Part BBa_K2534014 (RRE) gblock re-ligated to IGEM plasmid pS1BC3
Lane 14: Part BBa_K2534014 (RRE) gblock re-ligated to IGEM plasmid pS1BC3
Lane 15: DNA 1000 bp Ladder MW marker
Lane 1: DNA 100 bp Ladder MW marker
Lane 2: IGEM plasmid backbone pSB1C3
Lane 3: Part BBa_K2534016 (HIV LTR) ligated to pSB1C3
Lane 4: Part BBa_K2534020 (CCP/PPt) ligated to pSB1C3
Lane 5: Part BBa_K2534019 (HIV PSI) ligated to pSB1C3
Lane 6: Part BBa_K2534021 (Tet op) ligated to pSB1C3
3.miRNA Ligation to IGEM pS1BC3 Assembly after being synthesized using IDT offer
We then ligated our selected miRNA’s (miR-134) parts to the pS1BC3 backbone (figures 7-9).
Lane 1: DNA 100 bp Ladder MW marker
Lane 2: miR-134 ligated to IGEM plasmid pS1BC3
Lane 3: miR-370 ligated to IGEM plasmid pS1BC3
4.Competent E.coli Transformation
Our first transformations were a success! We managed to transform our competent cell colonies with our synthesized lentiviral transfer vector carrying our miRNA of interest (miR-134). With these cell we will be able to make more copies of each part in preparation for transformation (figures 8 & 9).
Transformation protocol
- Thaw competent cells on ice. - Chill approximately (15 μl) of the ligation mixture in a 1.5 ml microcentrifuge tube. - Add 50 µl of competent cells to the DNA. Mix gently by pipetting up and down or flicking the tube 4–5 times to mix the cells and DNA. Do not vortex. - Place the mixture on ice for 30 minutes. Do not mix. - Heat shock at 42°C for 90 seconds. Do not mix. - Add 950 µl of room temperature media* to the tube. - Place tube at 37°C for 2h. Shake vigorously (250 rpm) or rotate. - Warm selection plates to 37°C. - Spread 50–100 µl of the cells and ligation mixture onto the plates. - Incubate overnight at 37°C.
5.Transfection and Viability Results
48 h after transfection, marked decreases in RKO cell counts (figure 10) and viability (figure 11) were observed by inverted microscopy. All the experimental conditions induced a cell-death phenotype that could be easily distinguished from control, indicating cell death after transfection with our transfer vector carrying miR-134 vs. the empty vector backbone showing highly significant efficacy of our construct (p <0.001) (Table 1).
Transfection protocol
For mirRNA lentiviral transfection, RKO cells (2 × 105) were cultured in six-well plates for 24 h and transfected with miRNA transfer vector using Oligofectamine (Invitrogen) at the indicated concentrations. Cells were lysed after 30 h for RNA isolation and after 72 h for protein levels. shRNA lentivirus production and infection conditions for RKO cells were optimized in six-well plates. Cells were seeded at a density of 2 × 105 per well in a six-well plate, incubated for 24 h, infected using 1 mL of shRNA lentiviral supernatant, and incubated for 7 d and more using 2 μg/mL puromycin selection. Viral supernatants for all other cell lines were produced in RKO cells using a modified version of the three plasmid transfection protocols described. Briefly, RKO cells were transfected with a mixture of two helper plasmids, pVSV-G, packaging vectors, and the specific miRNA expressing vector along with Transit-293 lipid transfection reagent (Mirus). After 18 to 24 h, the medium was replaced, and after an additional 72h, supernatants containing viral particles were harvested, filtered through 0.45 μmol/L cellulose-acetate and filtrates frozen down at −80°C until use. Cells to be infected were plated in black-sided clear-bottomed 96-well plates at densities ranging from 500 to 4,000 cells per well depending on the particular cell type 18 to 24 before infection with virus. Infections were done in 200 μL of media supplemented with 8 μg/mL polybrene at 37°C and 5% CO2 for 4 h using between 0.1 and 2.0 μL of titer-normalized viral supernatant/well depending on the particular cell type. Medium was replaced 12 to 24 h later with 100 μL fresh medium and puromycin added to the wells after an additional 12 to 24 h (final concentration, 0.5-2.0 μg/mL)
Viability assay protocol
RKO cells were seeded into 96-well plates at a density of 3000 cell/well overnight. The next daycells were transfected using effectene (Qiagen) transfection reagent with P-EGFPn1 or 134-5pmiRNA at different concentration (2 or 3 µg) Cells were incubated at 37 °C for 72 h then the viability was assessed using the cell-titer blue kit (Promega). The viability was quantified as fluorescence intensity using a microplate reader, FLUOstar Omega (BMG LABTECH). Results are presented on a semi-log scale and represent the average of 3 biological replicates ± s.e.m. Asterisks denote statistical difference.
6.GFP and DAPI staining
We confirmed the transformation of the cells and their apoptotic features by measuring GFP fluorescence and DAPI staining of RKO cells. This was after transfection with miR-134 expression vector and Co-expression with the GFP expression vector in a ratio of 3:1. This was to compensate for any ligation errors in the auto-GFP expression by transfected plasmids. Apoptotic features were strongly represented as brightening DAPI indicating condensed DNA (figure 13).
