Team:HokkaidoU Japan/Protocols
From 2011.igem.org
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+ | </style></html> | ||
+ | =Experimental Procedures= | ||
+ | ==General Protocols== | ||
+ | ===Preparation of Competent cells (''E. coli'' DH5a)=== | ||
+ | '''Reagents''' | ||
- | + | '''TB''' (Transformation Buffer)(at 4C, filtration) | |
- | = | + | {| class="protocol" |
- | + | |reagents | |
+ | |amount | ||
+ | |Final concentration | ||
+ | |- | ||
+ | |1 M CaCl<sub>2</sub> (at RT, autoclaved) | ||
+ | |0.75 mL | ||
+ | |15 mM | ||
+ | |- | ||
+ | |4 M KCl (at RT, autoclaved) | ||
+ | |3.125 mL | ||
+ | |250 mM | ||
+ | |- | ||
+ | |1 M MnCl<sub>2</sub> (at 4C, autoclaved) | ||
+ | |2.75 mL | ||
+ | |55 mM | ||
+ | |- | ||
+ | |1 M PIPES (pH 6.7 by NaOH, at 4C, filtration) | ||
+ | |0.5 mL | ||
+ | |10 mM | ||
+ | |- | ||
+ | |Total | ||
+ | |50 mL | ||
+ | | | ||
+ | |} | ||
+ | '''Procedure''' | ||
+ | # Single colony isolation on LB plate | ||
+ | # Incubate the plate for 15-19 hrs at 37C | ||
+ | # Lift a colony into 2 mL of LB | ||
+ | # Culture cells at 37C for 12-16 hrs at 180-200 rpm | ||
+ | # Transfer 30 uL, 100 uL, 300 uL of the culture into 100 mL SOB medium, respectively | ||
+ | # Culture cells at 20C (for 24 hrs over) at 180-200 rpm (to ΔOD<sub>550nm</sub> = 0.5~0.6) | ||
+ | # Leave the 300 mL flask for 10 min on ice | ||
+ | # Transfer the culture into two 50 mL Falcon tube | ||
+ | # Centrifuge 7500 rpm at 4C for 20 min (TOMY TA-22 rotor), and discard sup | ||
+ | # Suspend the pellet in ice-cold 15 mL of TB (Transformation Buffer)(7.5 mL/tube) | ||
+ | # Centrifuge 7500 rpm at 4C for 2 min (TOMY TA-22 rotor), and discard sup | ||
+ | # Suspend the pellet in ice-cold 3.2 mL of TB | ||
+ | # Add 0.24 mL of DMSO (stirring, bit by bit) | ||
+ | # Leave the 50 mL Falcon tube for 10 min on ice | ||
+ | # Dispense 50 uL into 0.5 mL tube | ||
+ | # Freeze the suspension in liquid nitrogen | ||
+ | # Store at -80C | ||
- | {{:Team:HokkaidoU_Japan/footer}} | + | |
+ | ===Bacterial Transformations=== | ||
+ | # Add DNA solution to thawed competent cells | ||
+ | # Incubate the cells on ice for 30 min | ||
+ | # Heat shock the cells by immersion in a pre-heated water bath at 42C for 60 sec | ||
+ | # Incubate the cells on ice for 5 min | ||
+ | # Add 200 uL of SOB broth | ||
+ | # Incubate the cells at 37C for 2 hrs while the tubes are shaking | ||
+ | # Plate 200 uL of the transformation onto the dish | ||
+ | # Incubate the plate at 37C for 12-14 hrs | ||
+ | |||
+ | |||
+ | |||
+ | ===Mini-prep (Alkaline SDS Method)=== | ||
+ | |||
+ | '''Reagents''' | ||
+ | '''Solution I''' | ||
+ | (at RT, filtration 0.2 um, 50 mL) | ||
+ | {| class="protocol" | ||
+ | |- | ||
+ | |reagents | ||
+ | |amount | ||
+ | |Final concentration | ||
+ | |- | ||
+ | |Glucose (at RT) | ||
+ | |0.45 g | ||
+ | |50 mM | ||
+ | |- | ||
+ | |1 M Tris-HCl (pH8.0, at RT, autoclaved) | ||
+ | |1.25 mL | ||
+ | |25 mM | ||
+ | |- | ||
+ | |0.5 M EDTA (pH8.0, at RT, autoclaved) | ||
+ | |1 mL | ||
+ | |10 mM | ||
+ | |- | ||
+ | |Total | ||
+ | |50 mL | ||
+ | | | ||
+ | |} | ||
+ | <br> | ||
+ | |||
+ | '''Solution II''' | ||
+ | (at RT, filtration 0.2 um, 20 mL) | ||
+ | {| class="protocol" | ||
+ | |- | ||
+ | |reagents | ||
+ | |amount | ||
+ | |Final concentration | ||
+ | |- | ||
+ | |10 N NaOH (at RT) | ||
+ | |0.4 mL | ||
+ | |0.2 N | ||
+ | |- | ||
+ | |10% SDS (at RT, filtration) | ||
+ | |2 mL | ||
+ | |1% | ||
+ | |- | ||
+ | |Total | ||
+ | |20 mL | ||
+ | | | ||
+ | |} | ||
+ | <br> | ||
+ | |||
+ | '''Solution III''' | ||
+ | (at RT, filtration 0.2 um, 50 mL) | ||
+ | {| class="protocol" | ||
+ | |- | ||
+ | |reagents | ||
+ | |amount | ||
+ | |Final concentration | ||
+ | |- | ||
+ | |5 M CH<sub>3</sub>COOK | ||
+ | |30 mL | ||
+ | |3 M | ||
+ | |- | ||
+ | |CH<sub>3</sub>COOH | ||
+ | |5.75 mL | ||
+ | | | ||
+ | |- | ||
+ | |H<sub>2</sub>O | ||
+ | |14.25 mL | ||
+ | | | ||
+ | |- | ||
+ | |Total | ||
+ | |50 mL | ||
+ | | | ||
+ | |} | ||
+ | |||
+ | '''Procedure''' | ||
+ | # Lift colony ''E. coli'' into 2 mL LB contained antibiotics | ||
+ | # Culture cells at 37C for 16-20 hrs at 180-200 rpm | ||
+ | # Transfer 1.2-1.5 mL of culture into 1.5 mL tube | ||
+ | # Centrifuge the culture at 15,000 rpm for 1 min at 4C and discard sup | ||
+ | # Suspend the pellet in ice-cold 100 uL of Solution I | ||
+ | # Add 200 uL of Solution II to the suspension | ||
+ | # Mix by inverting the tube 10-20 times | ||
+ | # Add ice-cold 150 uL of Solution III to the suspension | ||
+ | # Mix by inverting the tube 10-20 times | ||
+ | # Leave the tube for 5 min on ice | ||
+ | # Add 10 uL of Chloroform | ||
+ | # Mix by inverting the tube 5-10 times | ||
+ | # Centrifuge the suspension at 15,000 rpm for 5 min at 4C | ||
+ | # Transfer the supernatant into new 1.5 mL tube↓ | ||
+ | # Add equal volume of isopropanol and mix by voltexing | ||
+ | # Leave the tube for 5 min at RT | ||
+ | # Centrifuge the suspension at 15,000 rpm for 10 min at 4C and discard sup | ||
+ | # Rinse the ppt by 70% EtOH and mix by voltexing | ||
+ | # Centrifuge the suspension at 15,000 rpm for 2 min at 4C and discard sup | ||
+ | # Dry up the ppt | ||
+ | # Dissolve the ppt in 50 uL of TE (pH 8.0) | ||
+ | # Add 1 uL of 10 mg/mL RNase A (4C and stock at –20C) | ||
+ | # Incubate for 30 min at 37C | ||
+ | # PCIAA and CIAA extraction | ||
+ | # Ethanol precipitation | ||
+ | # Dry up the ppt | ||
+ | # Dissolve the ppt in 50 uL of TE (pH 8.0) | ||
+ | |||
+ | |||
+ | |||
+ | |||
+ | ===PCR=== | ||
+ | |||
+ | '''Vector''' | ||
+ | '''Standard reaction setup''' | ||
+ | {|class="protocol" style="text-align:center;" | ||
+ | |- | ||
+ | !Component | ||
+ | !Volume | ||
+ | |- | ||
+ | |10x PCR Buffer | ||
+ | |5 uL | ||
+ | |- | ||
+ | |2mM dNTPs | ||
+ | |5 uL | ||
+ | |- | ||
+ | |25mM MgSO<sub>4</sub> | ||
+ | |3 uL | ||
+ | |- | ||
+ | |Suffix-F primer | ||
+ | |1 uL | ||
+ | |- | ||
+ | |Prefix-R primer | ||
+ | |1 uL | ||
+ | |- | ||
+ | |Template DNA | ||
+ | |1 uL | ||
+ | |- | ||
+ | |KOD -Plus- Neo | ||
+ | |1 uL | ||
+ | |- | ||
+ | |DW | ||
+ | |X uL | ||
+ | |- | ||
+ | |Total | ||
+ | |50 uL | ||
+ | |} | ||
+ | |||
+ | '''Cycling conditions ''' (2-step cycle) | ||
+ | {|class="protocol" | ||
+ | | Stage | ||
+ | | Temperature and Time | ||
+ | |- | ||
+ | | Predenature | ||
+ | | 94C 2 min | ||
+ | |- | ||
+ | | Denature | ||
+ | | 98C 10 sec | ||
+ | |- | ||
+ | | Extension | ||
+ | | 68C X sec (30 sec/kb) | ||
+ | |- | ||
+ | | Hold | ||
+ | | 4C | ||
+ | |} | ||
+ | * 30-40 cycles | ||
+ | '''Insert''' | ||
+ | '''Standard reaction setup''' | ||
+ | {|class="protocol" style="text-align:center;" | ||
+ | |- | ||
+ | !Component | ||
+ | !Volume | ||
+ | |- | ||
+ | |10x PCR Buffer | ||
+ | |5 uL | ||
+ | |- | ||
+ | |2mM dNTPs | ||
+ | |5 uL | ||
+ | |- | ||
+ | |25mM MgSO<sub>4</sub> | ||
+ | |3 uL | ||
+ | |- | ||
+ | |EX-F primer | ||
+ | |1 uL | ||
+ | |- | ||
+ | |PS-R primer | ||
+ | |1 uL | ||
+ | |- | ||
+ | |Template DNA | ||
+ | |1 uL | ||
+ | |- | ||
+ | |KOD -Plus- Neo | ||
+ | |1 uL | ||
+ | |- | ||
+ | |DW | ||
+ | |X uL | ||
+ | |- | ||
+ | |Total | ||
+ | |50 uL | ||
+ | |} | ||
+ | |||
+ | '''Cycling conditions''' (2-step cycle) | ||
+ | {|class="protocol" | ||
+ | | Stage | ||
+ | | Temperature and Time | ||
+ | |- | ||
+ | | Predenature | ||
+ | | 94C 2 min | ||
+ | |- | ||
+ | | Denature | ||
+ | | 98C 10 sec | ||
+ | |- | ||
+ | | Extension | ||
+ | | 68C X sec (30 sec/kb) | ||
+ | |- | ||
+ | | Hold | ||
+ | | 4C | ||
+ | |} | ||
+ | * 30-40 cycles | ||
+ | '''Colony PCR''' | ||
+ | * resuspend a colony into 10 uL of DW (template suspension) | ||
+ | * | ||
+ | '''Standard reaction setup''' | ||
+ | {|class="protocol" style="text-align:center;" | ||
+ | |- | ||
+ | !Component | ||
+ | !Volume | ||
+ | |- | ||
+ | |template suspension | ||
+ | |4.8 uL | ||
+ | |- | ||
+ | |Quick Taq | ||
+ | |5 uL | ||
+ | |- | ||
+ | |Forward primer | ||
+ | |0.1 uL | ||
+ | |- | ||
+ | |Reverse primer | ||
+ | |0.1 uL | ||
+ | |- | ||
+ | |Total | ||
+ | |10 uL | ||
+ | |} | ||
+ | |||
+ | '''Cycling conditions ''' (2-step cycle) | ||
+ | {|class="protocol" | ||
+ | | Stage | ||
+ | | Temperature and Time | ||
+ | |- | ||
+ | | Predenature | ||
+ | | 94C 2 min | ||
+ | |- | ||
+ | | Denature | ||
+ | | 94C 10 sec | ||
+ | |- | ||
+ | | Extension | ||
+ | | 68C X sec (60 sec/kb) | ||
+ | |- | ||
+ | | Hold | ||
+ | | 4C | ||
+ | |} | ||
+ | * 30-40 cycles | ||
+ | |||
+ | |||
+ | ===PCIAA and CIAA extraction=== | ||
+ | |||
+ | '''Reagent''' | ||
+ | * PCIAA = Phenol : CIAA = 1 : 1 | ||
+ | * CIAA = Chloroform : IsoAmyl Alcohol = 24 : 1 | ||
+ | |||
+ | '''Procedure''' | ||
+ | # Add equal volume of PCIAA and vortex vigorously | ||
+ | # Centrifuge at 15,000 rpm for 2 min at RT | ||
+ | # Transfer the aqueous phase to a new tube, being careful not to transfer the phase interface | ||
+ | # Add equal volume of CIAA and vortex vigorously | ||
+ | # Transfer the aqueous phase to a new tube | ||
+ | # Ethanol precipitation | ||
+ | |||
+ | |||
+ | |||
+ | |||
+ | |||
+ | ===Ethanol presipitation=== | ||
+ | |||
+ | # Add 1/10 volume of 3M CH<sub>3</sub>COONa | ||
+ | # Add 2.5 volume of 100% ethanol (EtOH) | ||
+ | # Incubate on ice for few min | ||
+ | # Centrifuge at 15,000 rpm for 10 min at 4C and discard sup | ||
+ | # Wash precipitation with 100 uL of 70% EtOH (EtOH has to be cold) | ||
+ | # Centrifuge at 15,000 rpm for 5 min at 4C and discard sup | ||
+ | # Dry up the ppt (no EtOH should be left) | ||
+ | # Resuspend ppt in wanted volume of TE | ||
+ | |||
+ | |||
+ | |||
+ | ===Mini-prep (QIAprep Spin Miniprep Kit)=== | ||
+ | |||
+ | # Resuspend pelleted bacterial cells in 250 µl Buffer P1 and transfer to a micro-centrifuge tube | ||
+ | # Add 250 µl Buffer P2 and mix thoroughly by inverting the tube 4–6 times | ||
+ | # Add 350 µl Buffer N3 and mix immediately and thoroughly by inverting the tube 4–6 times | ||
+ | # Centrifuge for 10 min at 13,000 rpm (~17,900 x g) in a table-top microcentrifuge | ||
+ | # Apply the supernatants from step 4 to the QIAprep spin column by decanting or pipetting | ||
+ | # Centrifuge for 30–60 s. Discard the flow-through | ||
+ | # Recommended: Wash the QIAprep spin column by adding 0.5 ml Buffer PB and centrifuging for 30–60 s. Discard the flow-through | ||
+ | # Wash QIAprep spin column by adding 0.75 ml Buffer PE and centrifuging for 30–60 s | ||
+ | # Discard the flow-through, and centrifuge for an additional 1 min to remove residual wash buffer | ||
+ | # Place the QIAprep column in a clean 1.5 ml microcentrifuge tube. To elute DNA, add 50 µl Buffer EB (10 mM Tris·Cl, pH 8.5) or water to the center of each QIAprep spin column, let stand for 1 min, and centrifuge for 1 min | ||
+ | |||
+ | * [http://www.qiagen.com/products/plasmid/qiaprepminiprepsystem/qiaprepspinminiprepkit.aspx see details (Official website)] | ||
+ | |||
+ | |||
+ | |||
+ | ===Gel Extraction (Wizard® SV Gel and PCR Clean-Up System)=== | ||
+ | |||
+ | # Following electrophoresis, excise DNA band from gel and place gel slice in a 1.5 mL microcentrifuge tube | ||
+ | # Add 10 uL Membrane Binding Solution per 10 mg of gel slice | ||
+ | # Vortex and incubate at 50–65C until gel slice is completely dissolved | ||
+ | # Insert SV Minicolumn into Collection Tube | ||
+ | # Transfer dissolved gel mixture or prepared PCR product to the Minicolumn assembly | ||
+ | # Incubate at room temperature for 1 min | ||
+ | # Centrifuge at 16,000 g for 1 min | ||
+ | # Discard flowthrough and reinsert Minicolumn into Collection Tube | ||
+ | # Add 700 uL Membrane Wash Solution (ethanol added) | ||
+ | # Centrifuge at 16,000 g for 1 min | ||
+ | # Discard flowthrough and reinsert Minicolumn into Collection Tube | ||
+ | # Repeat Step 4 with 500 uL Membrane Wash Solution | ||
+ | # Centrifuge at 16,000 g for 5 min | ||
+ | # Empty the Collection Tube and recentrifuge the column assembly for 1 min with the microcentrifuge lid open (or off) to allow evaporation of any residual ethanol | ||
+ | # Carefully transfer Minicolumn to a clean 1.5 mL microcentrifuge tube | ||
+ | # Add 50 uL of Nuclease-Free Water to the Minicolumn | ||
+ | # Incubate at room temperature for 1 min | ||
+ | # Centrifuge at 16,000 g for 1 min | ||
+ | # Discard Minicolumn and store DNA at 4C or –20C | ||
+ | * [http://www.promega.com/applications/pcr/featuresandbenefits/Wizard_SV_Gel_PCR_Clean-Up_System.htm see details (Official website)] | ||
+ | |||
+ | |||
+ | |||
+ | ==Infection assay== | ||
+ | ===Preparation of T3SS ''E. coli''=== | ||
+ | |||
+ | |||
+ | |||
+ | ===Infection assay using onion cells=== | ||
+ | |||
+ | |||
+ | |||
+ | ===Infection assay using HeLa cells=== | ||
+ | |||
+ | |||
+ | |||
+ | ===Detection of injected protein using GSK tag=== | ||
+ | |||
+ | '''Protein extraction from infected HeLa cells''' | ||
+ | |||
+ | '''SDS-PAGE and Western Blot analysis''' | ||
+ | |||
+ | HeLa cell lysates were subjected to SDS-PAGE, and separated proteins were transferred to an Immobilon-P membranes (Millipore). The membranes were blocked with Blocking buffer (20 mM Tris, 150 mM NaCl, 0.05% Tween 20, 5% nonfat milk) for 1 h at room temperature. The blots were probed with Phospho-GSK-3β (Ser9) antibody (Cell Signaling Technology #9336) or GSK-3β antibody (Cell Signaling Technology #9315) diluted 1/1000 in Blocking buffer and incubated overnight at room temperature. Blots were washed three times with TTBS (20 mM Tris, 150 mM NaCl, 0.05% Tween 20) for 15 min each time. Secondary antibody (alkaline phosphatase-conjugated anti-rabbit immunoglobulin G) was diluted 1/1000 in Blocking buffer and incubated with the blots for 1.5 h at 37C. Blots were washed as described above and developed with BCIP/NBT. | ||
+ | |||
+ | |||
+ | =Primers= | ||
+ | ===Note=== | ||
+ | {|class="primer" style="text-align:center;" | ||
+ | |- | ||
+ | !rowspan="2"|Primer Name | ||
+ | |colspan="4" style="text-align:left;"|Whole Sequence | ||
+ | |- | ||
+ | |style="width:61px;"|F/R||style="width:306px; text-align:left;"|Annealing Sequence||style="width:51px;"|Tm||style="width:306px; text-align:left;"|Adding Sequence | ||
+ | |} | ||
+ | |||
+ | |||
+ | ===General Primers=== | ||
+ | {|class="primer" style="text-align:center;" | ||
+ | |- | ||
+ | !rowspan="2"|EX_F | ||
+ | |colspan="4" style="text-align:left;"|gcagaattcgcggccgcttctagag | ||
+ | |- | ||
+ | |style="width:61px;"|Forward||style="width:306px; text-align:left"|Biobrick Prefix||style="width:51px;"|74.5 C||style="width:306px; text-align:left;"|None | ||
+ | |- | ||
+ | !rowspan="2"|PS_R | ||
+ | |colspan="4" style="text-align:left;"|agcctgcagcggccgctactagta | ||
+ | |- | ||
+ | |Reverse||style="text-align:left;"|Biobrick Suffix||74.6 C||style="text-align:left;"|None | ||
+ | |- | ||
+ | !rowspan="2"|suffix_F | ||
+ | |colspan="4" style="text-align:left;"|tactagtagcggccgctgcaggct | ||
+ | |- | ||
+ | |Forward||style="text-align:left;"|Biobrick Suffix||74.6 C||style="text-align:left;"|None | ||
+ | |- | ||
+ | !rowspan="2"|prefix_R | ||
+ | |colspan="4" style="text-align:left;"|ctctagaagcggccgcgaattctgc | ||
+ | |- | ||
+ | |Reverse||style="text-align:left;"|Biobrick Prefix||74.5 C||style="text-align:left;"|None | ||
+ | |- | ||
+ | !rowspan="2"|100up_EX_F | ||
+ | |colspan="4" style="text-align:left;"|aacctataaaaataccgcatacac | ||
+ | |- | ||
+ | |Forward||style="text-align:left;"|100bp upstream from Biobrick prefix||62.7 C||style="text-align:left;"|None | ||
+ | |- | ||
+ | !rowspan="2"|200dn_PS_R | ||
+ | |colspan="4" style="text-align:left;"|tcccctgattctgtggataaccgt | ||
+ | |- | ||
+ | |Reverse||style="text-align:left;"|200bp downstream from Biobrick suffix||66.6 C||style="text-align:left;"|None | ||
+ | |} | ||
+ | |||
+ | *[http://partsregistry.org/wiki/index.php?title=Part:BBa_K496000 See details of 100up_EX_F/200dn_PS_R] (partsregistry) | ||
+ | |||
+ | |||
+ | ===Primers Used for Backbone Construction=== | ||
+ | {|class="primer" style="text-align:center;" | ||
+ | |- | ||
+ | !rowspan="2"|EX_RBS_SlrP_F | ||
+ | |colspan="4" style="text-align:left;"|GCAGAATTCGCGGCCGCTTCTAGAaaagaggagaaaatatgtttaatattactaatatacaatctacggc | ||
+ | |- | ||
+ | |style="width:61px;"|Forward||style="width:306px; text-align:left"|RBS sequence, 5' terminal of SlrP coding sequence||style="width:51px"|69.7 C||style="width:306px; text-align:left;"|Biobrick Prefix | ||
+ | |- | ||
+ | !rowspan="2"|PS_SlrP_R | ||
+ | |colspan="4" style="text-align:left;"|AGCCTGCAGCGGCCGCTACTAGTggtaagtcctaatattttcagacgaag | ||
+ | |- | ||
+ | |Reverse||style="text-align:left;"|3'terminal of SlrP coding sequence||64.5 C||style="text-align:left;"|Biofusion Suffix | ||
+ | |- | ||
+ | !rowspan="2"|Bsa1_dt_F | ||
+ | |colspan="4" style="text-align:left;"|GGCGACTAGAGAGACCccaggcatcaaataaaacgaaag | ||
+ | |- | ||
+ | |Forward||style="text-align:left;"|5'terminal of double terminator sequence||63.6 C||style="text-align:left;"|BsaI recognition site and its cleavage site | ||
+ | |- | ||
+ | !rowspan="2"|Bsa1_SlrP_R | ||
+ | |colspan="4" style="text-align:left;"|GGCCTGGCCTGAGACCCCggtaagtcctaatattttcagacga | ||
+ | |- | ||
+ | |Reverse||style="text-align:left;"|3'terminal of SlrP coding Sequence||63.0 C||style="text-align:left;"|BsaI recognition site and its cleavage site | ||
+ | |- | ||
+ | !rowspan="2"|Bsa1_GSK_SlrP_R | ||
+ | |colspan="4" style="text-align:left;"|GGCCTGGCCTGAGACCCCACTTTCAGCGAAACTTGTAGTGCGAGGGCGACCACTCATggtaagtcctaatattttcagacga | ||
+ | |- | ||
+ | |Reverse||style="text-align:left;"|3'terminal of SlrP coding Sequence||63.6 C||style="text-align:left;"|GSK Tag, BsaI recognition site and its cleavage site | ||
+ | |} | ||
+ | |||
+ | *[https://2011.igem.org/Team:HokkaidoU_Japan/Project/Backbone See details of BsaI Backbone] (Project Page) | ||
+ | |||
+ | |||
+ | ===Sequencing Primer=== | ||
+ | {|class="primer" style="text-align:center;" | ||
+ | |- | ||
+ | !rowspan="2"|SlrP_373_to_394_F | ||
+ | |colspan="4" style="text-align:left;"|gaaagtcagtcacctatacccg | ||
+ | |- | ||
+ | |style="width:61px;"|Forward||style="width:306px; text-align:left;"|SlrP coding sequence, from 373bp to 394 bp||style="width:51px;"|63.4 C||style="width:306px; text-align:left;"|None | ||
+ | |} | ||
+ | |||
+ | {{Team:HokkaidoU_Japan/footer}} |
Revision as of 22:51, 3 October 2011
HokkaidoU Japan
iGEM 2011 Team of Hokkaido University
Contents |
Experimental Procedures
General Protocols
Preparation of Competent cells (E. coli DH5a)
Reagents
TB (Transformation Buffer)(at 4C, filtration)
reagents | amount | Final concentration |
1 M CaCl2 (at RT, autoclaved) | 0.75 mL | 15 mM |
4 M KCl (at RT, autoclaved) | 3.125 mL | 250 mM |
1 M MnCl2 (at 4C, autoclaved) | 2.75 mL | 55 mM |
1 M PIPES (pH 6.7 by NaOH, at 4C, filtration) | 0.5 mL | 10 mM |
Total | 50 mL |
Procedure
- Single colony isolation on LB plate
- Incubate the plate for 15-19 hrs at 37C
- Lift a colony into 2 mL of LB
- Culture cells at 37C for 12-16 hrs at 180-200 rpm
- Transfer 30 uL, 100 uL, 300 uL of the culture into 100 mL SOB medium, respectively
- Culture cells at 20C (for 24 hrs over) at 180-200 rpm (to ΔOD550nm = 0.5~0.6)
- Leave the 300 mL flask for 10 min on ice
- Transfer the culture into two 50 mL Falcon tube
- Centrifuge 7500 rpm at 4C for 20 min (TOMY TA-22 rotor), and discard sup
- Suspend the pellet in ice-cold 15 mL of TB (Transformation Buffer)(7.5 mL/tube)
- Centrifuge 7500 rpm at 4C for 2 min (TOMY TA-22 rotor), and discard sup
- Suspend the pellet in ice-cold 3.2 mL of TB
- Add 0.24 mL of DMSO (stirring, bit by bit)
- Leave the 50 mL Falcon tube for 10 min on ice
- Dispense 50 uL into 0.5 mL tube
- Freeze the suspension in liquid nitrogen
- Store at -80C
Bacterial Transformations
- Add DNA solution to thawed competent cells
- Incubate the cells on ice for 30 min
- Heat shock the cells by immersion in a pre-heated water bath at 42C for 60 sec
- Incubate the cells on ice for 5 min
- Add 200 uL of SOB broth
- Incubate the cells at 37C for 2 hrs while the tubes are shaking
- Plate 200 uL of the transformation onto the dish
- Incubate the plate at 37C for 12-14 hrs
Mini-prep (Alkaline SDS Method)
Reagents Solution I (at RT, filtration 0.2 um, 50 mL)
reagents | amount | Final concentration |
Glucose (at RT) | 0.45 g | 50 mM |
1 M Tris-HCl (pH8.0, at RT, autoclaved) | 1.25 mL | 25 mM |
0.5 M EDTA (pH8.0, at RT, autoclaved) | 1 mL | 10 mM |
Total | 50 mL |
Solution II (at RT, filtration 0.2 um, 20 mL)
reagents | amount | Final concentration |
10 N NaOH (at RT) | 0.4 mL | 0.2 N |
10% SDS (at RT, filtration) | 2 mL | 1% |
Total | 20 mL |
Solution III (at RT, filtration 0.2 um, 50 mL)
reagents | amount | Final concentration |
5 M CH3COOK | 30 mL | 3 M |
CH3COOH | 5.75 mL | |
H2O | 14.25 mL | |
Total | 50 mL |
Procedure
- Lift colony E. coli into 2 mL LB contained antibiotics
- Culture cells at 37C for 16-20 hrs at 180-200 rpm
- Transfer 1.2-1.5 mL of culture into 1.5 mL tube
- Centrifuge the culture at 15,000 rpm for 1 min at 4C and discard sup
- Suspend the pellet in ice-cold 100 uL of Solution I
- Add 200 uL of Solution II to the suspension
- Mix by inverting the tube 10-20 times
- Add ice-cold 150 uL of Solution III to the suspension
- Mix by inverting the tube 10-20 times
- Leave the tube for 5 min on ice
- Add 10 uL of Chloroform
- Mix by inverting the tube 5-10 times
- Centrifuge the suspension at 15,000 rpm for 5 min at 4C
- Transfer the supernatant into new 1.5 mL tube↓
- Add equal volume of isopropanol and mix by voltexing
- Leave the tube for 5 min at RT
- Centrifuge the suspension at 15,000 rpm for 10 min at 4C and discard sup
- Rinse the ppt by 70% EtOH and mix by voltexing
- Centrifuge the suspension at 15,000 rpm for 2 min at 4C and discard sup
- Dry up the ppt
- Dissolve the ppt in 50 uL of TE (pH 8.0)
- Add 1 uL of 10 mg/mL RNase A (4C and stock at –20C)
- Incubate for 30 min at 37C
- PCIAA and CIAA extraction
- Ethanol precipitation
- Dry up the ppt
- Dissolve the ppt in 50 uL of TE (pH 8.0)
PCR
Vector Standard reaction setup
Component | Volume |
---|---|
10x PCR Buffer | 5 uL |
2mM dNTPs | 5 uL |
25mM MgSO4 | 3 uL |
Suffix-F primer | 1 uL |
Prefix-R primer | 1 uL |
Template DNA | 1 uL |
KOD -Plus- Neo | 1 uL |
DW | X uL |
Total | 50 uL |
Cycling conditions (2-step cycle)
Stage | Temperature and Time |
Predenature | 94C 2 min |
Denature | 98C 10 sec |
Extension | 68C X sec (30 sec/kb) |
Hold | 4C |
- 30-40 cycles
Insert Standard reaction setup
Component | Volume |
---|---|
10x PCR Buffer | 5 uL |
2mM dNTPs | 5 uL |
25mM MgSO4 | 3 uL |
EX-F primer | 1 uL |
PS-R primer | 1 uL |
Template DNA | 1 uL |
KOD -Plus- Neo | 1 uL |
DW | X uL |
Total | 50 uL |
Cycling conditions (2-step cycle)
Stage | Temperature and Time |
Predenature | 94C 2 min |
Denature | 98C 10 sec |
Extension | 68C X sec (30 sec/kb) |
Hold | 4C |
- 30-40 cycles
Colony PCR
- resuspend a colony into 10 uL of DW (template suspension)
Standard reaction setup
Component | Volume |
---|---|
template suspension | 4.8 uL |
Quick Taq | 5 uL |
Forward primer | 0.1 uL |
Reverse primer | 0.1 uL |
Total | 10 uL |
Cycling conditions (2-step cycle)
Stage | Temperature and Time |
Predenature | 94C 2 min |
Denature | 94C 10 sec |
Extension | 68C X sec (60 sec/kb) |
Hold | 4C |
- 30-40 cycles
PCIAA and CIAA extraction
Reagent
- PCIAA = Phenol : CIAA = 1 : 1
- CIAA = Chloroform : IsoAmyl Alcohol = 24 : 1
Procedure
- Add equal volume of PCIAA and vortex vigorously
- Centrifuge at 15,000 rpm for 2 min at RT
- Transfer the aqueous phase to a new tube, being careful not to transfer the phase interface
- Add equal volume of CIAA and vortex vigorously
- Transfer the aqueous phase to a new tube
- Ethanol precipitation
Ethanol presipitation
- Add 1/10 volume of 3M CH3COONa
- Add 2.5 volume of 100% ethanol (EtOH)
- Incubate on ice for few min
- Centrifuge at 15,000 rpm for 10 min at 4C and discard sup
- Wash precipitation with 100 uL of 70% EtOH (EtOH has to be cold)
- Centrifuge at 15,000 rpm for 5 min at 4C and discard sup
- Dry up the ppt (no EtOH should be left)
- Resuspend ppt in wanted volume of TE
Mini-prep (QIAprep Spin Miniprep Kit)
- Resuspend pelleted bacterial cells in 250 µl Buffer P1 and transfer to a micro-centrifuge tube
- Add 250 µl Buffer P2 and mix thoroughly by inverting the tube 4–6 times
- Add 350 µl Buffer N3 and mix immediately and thoroughly by inverting the tube 4–6 times
- Centrifuge for 10 min at 13,000 rpm (~17,900 x g) in a table-top microcentrifuge
- Apply the supernatants from step 4 to the QIAprep spin column by decanting or pipetting
- Centrifuge for 30–60 s. Discard the flow-through
- Recommended: Wash the QIAprep spin column by adding 0.5 ml Buffer PB and centrifuging for 30–60 s. Discard the flow-through
- Wash QIAprep spin column by adding 0.75 ml Buffer PE and centrifuging for 30–60 s
- Discard the flow-through, and centrifuge for an additional 1 min to remove residual wash buffer
- Place the QIAprep column in a clean 1.5 ml microcentrifuge tube. To elute DNA, add 50 µl Buffer EB (10 mM Tris·Cl, pH 8.5) or water to the center of each QIAprep spin column, let stand for 1 min, and centrifuge for 1 min
- [http://www.qiagen.com/products/plasmid/qiaprepminiprepsystem/qiaprepspinminiprepkit.aspx see details (Official website)]
Gel Extraction (Wizard® SV Gel and PCR Clean-Up System)
- Following electrophoresis, excise DNA band from gel and place gel slice in a 1.5 mL microcentrifuge tube
- Add 10 uL Membrane Binding Solution per 10 mg of gel slice
- Vortex and incubate at 50–65C until gel slice is completely dissolved
- Insert SV Minicolumn into Collection Tube
- Transfer dissolved gel mixture or prepared PCR product to the Minicolumn assembly
- Incubate at room temperature for 1 min
- Centrifuge at 16,000 g for 1 min
- Discard flowthrough and reinsert Minicolumn into Collection Tube
- Add 700 uL Membrane Wash Solution (ethanol added)
- Centrifuge at 16,000 g for 1 min
- Discard flowthrough and reinsert Minicolumn into Collection Tube
- Repeat Step 4 with 500 uL Membrane Wash Solution
- Centrifuge at 16,000 g for 5 min
- Empty the Collection Tube and recentrifuge the column assembly for 1 min with the microcentrifuge lid open (or off) to allow evaporation of any residual ethanol
- Carefully transfer Minicolumn to a clean 1.5 mL microcentrifuge tube
- Add 50 uL of Nuclease-Free Water to the Minicolumn
- Incubate at room temperature for 1 min
- Centrifuge at 16,000 g for 1 min
- Discard Minicolumn and store DNA at 4C or –20C
- [http://www.promega.com/applications/pcr/featuresandbenefits/Wizard_SV_Gel_PCR_Clean-Up_System.htm see details (Official website)]
Infection assay
Preparation of T3SS E. coli
Infection assay using onion cells
Infection assay using HeLa cells
Detection of injected protein using GSK tag
Protein extraction from infected HeLa cells
SDS-PAGE and Western Blot analysis
HeLa cell lysates were subjected to SDS-PAGE, and separated proteins were transferred to an Immobilon-P membranes (Millipore). The membranes were blocked with Blocking buffer (20 mM Tris, 150 mM NaCl, 0.05% Tween 20, 5% nonfat milk) for 1 h at room temperature. The blots were probed with Phospho-GSK-3β (Ser9) antibody (Cell Signaling Technology #9336) or GSK-3β antibody (Cell Signaling Technology #9315) diluted 1/1000 in Blocking buffer and incubated overnight at room temperature. Blots were washed three times with TTBS (20 mM Tris, 150 mM NaCl, 0.05% Tween 20) for 15 min each time. Secondary antibody (alkaline phosphatase-conjugated anti-rabbit immunoglobulin G) was diluted 1/1000 in Blocking buffer and incubated with the blots for 1.5 h at 37C. Blots were washed as described above and developed with BCIP/NBT.
Primers
Note
Primer Name | Whole Sequence | |||
---|---|---|---|---|
F/R | Annealing Sequence | Tm | Adding Sequence |
General Primers
EX_F | gcagaattcgcggccgcttctagag | |||
---|---|---|---|---|
Forward | Biobrick Prefix | 74.5 C | None | |
PS_R | agcctgcagcggccgctactagta | |||
Reverse | Biobrick Suffix | 74.6 C | None | |
suffix_F | tactagtagcggccgctgcaggct | |||
Forward | Biobrick Suffix | 74.6 C | None | |
prefix_R | ctctagaagcggccgcgaattctgc | |||
Reverse | Biobrick Prefix | 74.5 C | None | |
100up_EX_F | aacctataaaaataccgcatacac | |||
Forward | 100bp upstream from Biobrick prefix | 62.7 C | None | |
200dn_PS_R | tcccctgattctgtggataaccgt | |||
Reverse | 200bp downstream from Biobrick suffix | 66.6 C | None |
- [http://partsregistry.org/wiki/index.php?title=Part:BBa_K496000 See details of 100up_EX_F/200dn_PS_R] (partsregistry)
Primers Used for Backbone Construction
EX_RBS_SlrP_F | GCAGAATTCGCGGCCGCTTCTAGAaaagaggagaaaatatgtttaatattactaatatacaatctacggc | |||
---|---|---|---|---|
Forward | RBS sequence, 5' terminal of SlrP coding sequence | 69.7 C | Biobrick Prefix | |
PS_SlrP_R | AGCCTGCAGCGGCCGCTACTAGTggtaagtcctaatattttcagacgaag | |||
Reverse | 3'terminal of SlrP coding sequence | 64.5 C | Biofusion Suffix | |
Bsa1_dt_F | GGCGACTAGAGAGACCccaggcatcaaataaaacgaaag | |||
Forward | 5'terminal of double terminator sequence | 63.6 C | BsaI recognition site and its cleavage site | |
Bsa1_SlrP_R | GGCCTGGCCTGAGACCCCggtaagtcctaatattttcagacga | |||
Reverse | 3'terminal of SlrP coding Sequence | 63.0 C | BsaI recognition site and its cleavage site | |
Bsa1_GSK_SlrP_R | GGCCTGGCCTGAGACCCCACTTTCAGCGAAACTTGTAGTGCGAGGGCGACCACTCATggtaagtcctaatattttcagacga | |||
Reverse | 3'terminal of SlrP coding Sequence | 63.6 C | GSK Tag, BsaI recognition site and its cleavage site |
- See details of BsaI Backbone (Project Page)
Sequencing Primer
SlrP_373_to_394_F | gaaagtcagtcacctatacccg | |||
---|---|---|---|---|
Forward | SlrP coding sequence, from 373bp to 394 bp | 63.4 C | None |