TITLE: 6L Live PlmCasX Expression & Purification
AUTHORS: Liz O'Brien, Connor Tsuchida

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This protocol describes methods to express and purify PlmCasX plasmids.</div></div>

[STEPS]
?. [Day 1: Grow pre-culture]
Set up pre-culture by either transforming plasmid into Rosetta cells or using a small amount from a glycerol stock (steps found below).
Perform all steps aseptically, next to a Bunsen burner.
?. [Day 1: Grow pre-culture]
Add  to a 500 ml baffled Erlenmeyer flask.
[LB broth]
?. [Day 1: Grow pre-culture]
Add Ampicillin to a final concentration of  (add  of  Ampicillin).
200 µl
?. [Day 1: Grow pre-culture]
Add the entire tube of transformed cells to flask.
?. [Day 1: Grow pre-culture]
Shake at ,  – , .
37 °C
Centrifuge: 160 33
Centrifuge: 180 33
?. [Day 2: Grow TB culture]
Locate 6 glass or plastic baffled 1 l flasks and add  to each flask.
[TB broth]
Perform all steps in this section aseptically, next to a Bunsen burner.
?. [Day 2: Grow TB culture]
Add Ampicillin to a final concentration of  (add  of  amp per flask).
1 ml
?. [Day 2: Grow TB culture]
Add  –  of pre-culture (grown in LB broth) to each flask of TB broth.
20 ml
25 ml
?. [Day 2: Grow TB culture]
Shake at ,  – until the OD600 measurement is between 0.5 – 0.6 (roughly 3 – 6 hours).
37 °C
Centrifuge: 160 33
Centrifuge: 180 33
Be sure to blank spectrophotometer with cuvette of clean media.
?. [Day 2: Induce protein expression]
Transfer TB flasks to be .
on ice
?. [Day 2: Induce protein expression]
Add IPTG to a final concentration of  to each flask ( IPTG per flask).
1 ml
?. [Day 2: Induce protein expression]
Shake at ,  – , .
16 °C
Centrifuge: 160 33
Centrifuge: 180 33
?. [Day 3: Lyse bacterial cells]
Transfer cultures to 1L plastic centrifuge tubes and balance pairs of tubes by adding MilliQ H2O.
?. [Day 3: Lyse bacterial cells]
Centrifuge cultures in a Beckman Coulter J6-MI centrifuge at  ,  ,  .
Centrifuge: 4000 33
4 °C
Use blue sleeves to ensure tubes fit snuggly.
?. [Day 3: Lyse bacterial cells]
Pour off supernatant from cultures.
?. [Day 3: Lyse bacterial cells]
Add  Lysis Buffer + TCEP to each plastic tube.
20 ml
?. [Day 3: Lyse bacterial cells]
Vortex tubes until sample is homogenous.
Use a flat-top vortex.
?. [Day 3: Lyse bacterial cells]
Transfer cell suspension to a 500 ml beaker.
Alternatively, lysed cells can be frozen down for later use. Transfer to 5mL tubes and centrifuge at 4,000rpm, 20 minutes, 4°C. Pour off supernatant and freeze pellet at -20°C.
?. [Day 3: Lyse bacterial cells]
Add solution of tablets + PMSF + DNase to cell suspension.
?. [Day 3: Lyse bacterial cells]
Place sample in tube of ice with water to hold beaker snugly.
?. [Day 3: Lyse bacterial cells]
Sonicate sample in cold room.
?. [Day 3: Lyse bacterial cells]
Rinse tip of sonicator with ethanol, then H2O.
?. [Day 3: Lyse bacterial cells]
Set sonicator to run at 5.5 power setting for , cycling between  and .
[on]
[off]
?. [Day 3: Lyse bacterial cells]
Place 6 plastic ultracentrifuge tubes (with caps) .
on ice
?. [Day 3: Lyse bacterial cells]
Divide sonicated sample between ultracentrifuge tubes. Use scale to match pairs within +/- 0.01 g by adding MilliQ H2O.
?. [Day 3: Lyse bacterial cells]
Centrifuge samples in Beckman Coulter Avanti J-E centrifuge at , , .
Centrifuge: 35000 33
4 °C
?. [Day 3: Lyse bacterial cells]
Filter sonicated sample through bottle top 0.22 μm filter and store .
on ice
?. [Day 3: Ni-NTA column binding]
Add  Qiagen Ni-NTA resin to yellow gravity column (Econo-Column Chromatography Column) with butterfly valve in  cold room.
20 ml
4 °C
?. [Day 3: Ni-NTA column binding]
Drain off ethanol storage buffer.
?. [Day 3: Ni-NTA column binding]
Wash with  Lysis Buffer + TCEP. (Repeat 4 more times).
10 ml
?. [Day 3: Ni-NTA column binding]
Add  Lysis Buffer + TCEP to Ni-NTA column, mix with pipette.
10 ml
?. [Day 3: Ni-NTA column binding]
Transfer Ni-NTA resin to bottle with sonicated/filtered sample and add stir bar.
Do not discard yellow gravity column, it will be used again in step 33.
?. [Day 3: Ni-NTA column binding]
Stir sample in Ni-NTA resin at  for at least .
4 °C
?. [Day 3: Ni-NTA column binding]
Add sample in Ni-NTA resin back to yellow gravity column.
?. [Day 3: Ni-NTA column binding]
Drain liquid.
Collect flow-through for gel electrophoresis.
?. [Day 3: Ni-NTA column binding]
Add  Lysis Buffer + TCEP.
50 ml
?. [Day 3: Ni-NTA column binding]
Drain liquid.
Collect flow-through for gel electrophoresis.
?. [Day 3: Ni-NTA column binding]
Add  Lysis Buffer + TCEP.
10 ml
?. [Day 3: Ni-NTA column binding]
Add  TEV protease (4 eppendorfs of  lab stock).
250 µl
?. [Day 3: Ni-NTA column binding]
Mix sample in Ni-NTA resin well.
?. [Day 3: Ni-NTA column binding]
Incubate column at , .
4 °C
?. [Day 4: Elution of Ni-NTA column]
Elute Ni-NTA column into 50 ml Falcon tube. Keep . Label as “flow-through.”
on ice
?. [Day 4: Elution of Ni-NTA column]
Wash column with  Elution Buffer (). (Repeat this step 2 more times). Collect samples into 50 ml Falcon tubes and label with Elution numbers (I.e. label elution #1 this step, elution #2 next, etc.).
10 ml
[imidazole]
?. [Day 4: Elution of Ni-NTA column]
Wash column with  Elution Buffer ().
10 ml
[imidazole]
?. [Day 4: Elution of Ni-NTA column]
Wash column with  Elution Buffer ().
10 ml
[imidazole]
?. [Day 3: Ni-NTA column binding]
Wash with  Lysis Buffer + TCEP.
10 ml
?. [Day 3: Ni-NTA column binding]
Wash with  Lysis Buffer + TCEP.
10 ml
?. [Day 3: Ni-NTA column binding]
Wash with  Lysis Buffer + TCEP.
10 ml
?. [Day 3: Ni-NTA column binding]
Wash with  Lysis Buffer + TCEP.
10 ml
?. [Day 4: Elution of Ni-NTA column]
Pool fractions into 15 ml 50 kDa MWCO Amicon Concentrators.
?. [Day 4: Elution of Ni-NTA column]
Centrifuge at ,  until volume is less than .
Centrifuge: 4000 33
4 °C
4 ml
?. [Day 4: Elution of Ni-NTA column]
Transfer to 4 ml 30 kDa MWCO Amicon Concentrator.
?. [Day 4: Elution of Ni-NTA column]
Centrifuge until volume is less than .
500 µl
?. [Day 4: SP Column Ion Exchange]
Equilibrate column with  IEX A Buffer.
40 ml
Run 5 – 10 Column Volumes at 1 –  5 ml/min, using pre-column pressure alarm set to 0.5 MPa.
?. [Day 4: SP Column Ion Exchange]
Run Äkta at 0.25 ml/min to detach column.
?. [Day 4: SP Column Ion Exchange]
Draw up  sample with a 1 ml syringe.
500 µl
?. [Day 4: SP Column Ion Exchange]
Attach threaded luer lock attachment and remove air from syringe.
?. [Day 4: SP Column Ion Exchange]
Insert new 96 deep well plate into position 3.
?. [Day 4: SP Column Ion Exchange]
Run SP column gradient program between IEX Buffer A and B to elute.
?. [Day 4: SP Column Ion Exchange]
Collect fractions from chromatogram peak. Concentrate fractions if necessary.
Save samples for gel electrophoresis.
?. [Day 5: Size Exclusion Chromatography]
Run Äkta at 0.25 ml/min to attached S200 10/300 increase column.
Use 24 ml column, run at 0.5 ml/min, with 2.8 MPa pre-column pressure.
?. [Day 4: SP Column Ion Exchange]
Manually inject sample into top of 5 ml SP column.
Use wet connections and don’t exceed 5 ml/min.
?. [Day 4: SP Column Ion Exchange]
Run Äkta at 0.25 ml/min to attach column.
?. [Day 5: Size Exclusion Chromatography]
Add  loop to Äkta.
500 µl
?. [Day 5: Size Exclusion Chromatography]
Inject  SEC Buffer into loop with 5 ml syringe with the red luer lock attachment.
3 ml
?. [Day 5: Size Exclusion Chromatography]
Draw up sample into 1 ml syringe with the red luer lock attachment.
?. [Day 5: Size Exclusion Chromatography]
Inject sample (which should be less than ) into loop, leaving syringe attached.
500 µl
?. [Day 5: Size Exclusion Chromatography]
Run SEC program. The sample should be injected from the loop.
In System Control on the Akta software, go to the menu bar at the top of the page, select Method Editor > New.  Under column type, select Gel Filtration.  Choose ‘Superdex 200 10/300 Increase’ for the Column type.
?. [Day 5: Size Exclusion Chromatography]
Collect fractions from chromatogram. Concentrate as necessary.
Save samples for gel.
?. [Day 5: Size Exclusion Chromatography]
Aliquot into tubes.
?. [Day 5: Size Exclusion Chromatography]
Flash freeze in liquid nitrogen and store at  .
-80 °C
?. [Day 5: Size Exclusion Chromatography]
Nanodrop protein amount.
?. [Day 1: Grow pre-culture]
Add  to a 500 ml baffled Erlenmeyer flask.
[LB broth]
?. [Day 1: Grow pre-culture]
Add Ampicillin to a final concentration of  (add  of ).
200 µl
?. [Day 1: Grow pre-culture]
Add the entire tube of transformed cells to flask.
?. [Day 1: Grow pre-culture]
Shake at ,  – , .
37 °C
Centrifuge: 160 33
Centrifuge: 180 33
?. [Day 2: Grow TB culture]
Locate 6 glass or plastic baffled 1 l flasks and add  to each flask.
Do entire steps of this section near Bunsen burner.
[TB broth]
?. [Day 2: Grow TB culture]
Add Ampicillin to a final concentration of  (add  of  amp per flask).
1 ml
?. [Day 2: Grow TB culture]
Add  –  of pre-culture (grown in LB broth) to each flask of TB broth.
20 ml
25 ml
?. [Day 2: Grow TB culture]
Shake at ,  – until OD600 measurement is between 0.5 – 0.6 (roughly 3 – 6 hours).
37 °C
Centrifuge: 160 33
Centrifuge: 180 33
Be sure to blank spectrophotometer with cuvette of clean media.
?. [Day 2: Induce protein expression]
Transfer TB flasks to be .
on ice
?. [Day 2: Induce protein expression]
Add IPTG to a final concentration of  to each flask ( IPTG per flask).
1 ml
?. [Day 2: Induce protein expression]
Shake at ,  – , .
16 °C
Centrifuge: 160 33
Centrifuge: 180 33
?. [Day 3: Lyse cells]
Transfer cultures to 1 l plastic centrifuge tubes and balance pairs of tubes by adding MilliQ H2O.
?. [Day 3: Lyse cells]
Centrifuge cultures in a Beckman Coulter J6-MI centrifuge at  ,  ,  .
Centrifuge: 4000 33
4 °C
Use blue sleeves to ensure tubes fit snuggly.
?. [Day 3: Lyse cells]
Pour off supernatant from cultures.
?. [Day 3: Lyse cells]
Add  Lysis Buffer + TCEP to each plastic tube.
20 ml
?. [Day 3: Lyse cells]
Vortex tubes until sample is homogenous.
Use a flat-top vortex.
?. [Day 3: Lyse cells]
Transfer cell suspension to a 500 ml beaker.
Alternatively, lysed cells can be frozen down for later use. Transfer to 5mL tubes and centrifuge at 4,000rpm, 20 minutes, 4°C. Pour off supernatant and freeze pellet at -20°C.
?. [Day 3: Lyse cells]
In a separate beaker, mix together the following reagents:
?. [Day 3: Lyse cells]
Add  Lysis Buffer + TCEP.
10 ml
?. [Day 3: Lyse cells]
Add 8 tablets of ROCHE cOmplete tablets (1 tablet per 20 ml).
?. [Day 3: Lyse cells]
Add PMSF to final concentration  ( of  stock PMSF).
800 µl
?. [Day 3: Lyse cells]
Add  RNase-free DNase (2 μl per 20 ml).
8 µl
?. [Day 3: Lyse cells]
Add solution of tablets + PMSF + DNase to cell suspension.
?. [Day 3: Lyse cells]
Place sample in tube of ice with water to hold beaker snuggly.
?. [Day 3: Lyse cells]
Sonicate sample in cold room.
?. [Day 3: Lyse cells]
Rinse tip of sonicator with ethanol, then H2O.
?. [Day 3: Lyse cells]
Set sonicator to run at 5.5 power setting for , cycling between  and .
[on]
[off]
?. [Day 3: Lyse cells]
Place 6 plastic ultracentrifuge tubes (with caps) .
on ice
?. [Day 3: Lyse cells]
Divide sonicated sample between ultracentrifuge tubes. Use scale to match pairs within +/- 0.01 g by adding MilliQ H2O.
?. [Day 3: Lyse cells]
Centrifuge samples in Beckman Coulter Avanti J-E centrifuge at , , .
Centrifuge: 35000 33
4 °C
?. [Day 3: Lyse cells]
Filter sonicated sample through bottle top 0.22 μm filter and store .
on ice
?. [Day 3: Ni-NTA column binding]
Add  Qiagen Ni-NTA resin to yellow gravity column with butterfly valve in  cold room.
20 ml
4 °C
?. [Day 3: Ni-NTA column binding]
Drain off ethanol storage buffer.
?. [Day 3: Ni-NTA column binding]
Wash with  Lysis Buffer + TCEP. (Repeat 4 more times).
10 ml
?. [Day 3: Ni-NTA column binding]
Wash with  Lysis Buffer + TCEP.
10 ml
?. [Day 3: Ni-NTA column binding]
Wash with  Lysis Buffer + TCEP.
10 ml
?. [Day 3: Ni-NTA column binding]
Wash with  Lysis Buffer + TCEP.
10 ml
?. [Day 3: Ni-NTA column binding]
Wash with  Lysis Buffer + TCEP.
10 ml
?. [Day 3: Ni-NTA column binding]
Add  Lysis Buffer + TCEP to Ni-NTA column, mix with pipette.
10 ml
?. [Day 3: Ni-NTA column binding]
Transfer Ni-NTA resin to bottle with sonicated/filtered sample and add stir bar.
Do not discard yellow gravity column, it will be used again in step 33.
?. [Day 3: Ni-NTA column binding]
Stir sample in Ni-NTA resin at  for at least .
4 °C
?. [Day 3: Ni-NTA column binding]
Add sample in Ni-NTA resin back to yellow gravity column.
?. [Day 3: Ni-NTA column binding]
Drain liquid.
Collect flow-through for gel electrophoresis.
?. [Day 3: Ni-NTA column binding]
Add  Lysis Buffer + TCEP.
50 ml
?. [Day 3: Ni-NTA column binding]
Drain liquid.
Collect flow-through for gel electrophoresis.
?. [Day 3: Ni-NTA column binding]
Add  Lysis Buffer + TCEP.
10 ml
?. [Day 3: Ni-NTA column binding]
Add TEV protease (4 eppendorfs of high conc. lab stock).
?. [Day 3: Ni-NTA column binding]
Mix sample in Ni-NTA resin well.
?. [Day 3: Ni-NTA column binding]
Incubate column at , .
4 °C
?. [Day 4: Elution of Ni-NTA column]
Elute Ni-NTA column into 50 ml Falcon tube. Keep . Label as “flow-through.”
on ice
?. [Day 4: Elution of Ni-NTA column]
Wash column with  Elution Buffer (). (Repeat this step 2 more times). Collect samples into 50 ml Falcon tubes and label with Elution numbers (I.e. label elution #1 this step, elution #2 next, etc.).
10 ml
[imidazole]
?. [Day 4: Elution of Ni-NTA column]
Wash column with  Elution Buffer ().
10 ml
[imidazole]
?. [Day 4: Elution of Ni-NTA column]
Wash column with  Elution Buffer ().
10 ml
[imidazole]
?. [Day 4: Elution of Ni-NTA column]
Pool fractions into 15 ml 50 kDa MWCO Amicon Concentrators.
?. [Day 4: Elution of Ni-NTA column]
Centrifuge at ,  until volume is less than .
Centrifuge: 4000 33
4 °C
4 ml
?. [Day 4: Elution of Ni-NTA column]
Transfer to 4 ml 30 kDa MWCO Amicon Concentrator.
?. [Day 4: Elution of Ni-NTA column]
Centrifuge until volume is less than .
500 µl
?. [Day 4: SP Column Ion Exchange]
Run Äkta at 0.25 ml/min to attach column.
Use a 5 ml SP Column, Screw the bottom of column into top Äkta port, making sure to use wet connections. Use pre-column pressure alarm set at 0.5 MPa.
?. [Day 4: SP Column Ion Exchange]
Equilibrate column with  IEX A Buffer.
40 ml
Run 5 – 10 Column Volumes at 1 –  5 ml/min, using pre-column pressure alarm set to 0.5 MPa.
?. [Day 4: SP Column Ion Exchange]
Run Äkta at 0.25 ml/min to detach column.
?. [Day 4: SP Column Ion Exchange]
Draw up  sample with a 1 ml syringe.
500 µl
?. [Day 4: SP Column Ion Exchange]
Attach threaded luer lock attachment and remove air from syringe.
?. [Day 4: SP Column Ion Exchange]
Manually inject sample into top of 5 ml SP column.
Use wet connections and don’t exceed 5 ml/min.
?. [Day 4: SP Column Ion Exchange]
Run Äkta at 0.25 ml/min to attach column.
?. [Day 4: SP Column Ion Exchange]
Insert new 96 deep well plate into position 3.
?. [Day 4: SP Column Ion Exchange]
Run SP column gradient program between IEX Buffer A and B to elute.
?. [Day 4: SP Column Ion Exchange]
Collect fractions from chromatogram peak. Concentrate fractions if necessary.
Save samples for gel electrophoresis.
?. [Day 5: Size Exclusion Chromatography]
Run Äkta at 0.25 ml/min to attached S200 10/300 increase column.
Use 24 ml column, run at 0.5 ml/min, with 2.8 MPa pre-column pressure.
?. [Day 5: Size Exclusion Chromatography]
Add  loop to Äkta.
500 µl
?. [Day 5: Size Exclusion Chromatography]
Inject  SEC Buffer into loop with 5 ml syringe with the red luer lock attachment.
3 ml
?. [Day 5: Size Exclusion Chromatography]
Draw up sample into 1 ml syringe with the red luer lock attachment.
?. [Day 5: Size Exclusion Chromatography]
Inject sample (which should be less than ) into loop, leaving syringe attached.
500 µl
?. [Day 5: Size Exclusion Chromatography]
Run SEC program.
Should inject sample from loop.
?. [Day 5: Size Exclusion Chromatography]
Collect fractions from chromatogram. Concentrate as necessary.
Save samples for gel.
?. [Day 5: Size Exclusion Chromatography]
Nanodrop protein amount.
?. [Day 5: Size Exclusion Chromatography]
Aliquot into tubes.
?. [Day 5: Size Exclusion Chromatography]
Flash freeze in liquid nitrogen and store at  .
-80 °C
?. [Day 3: Lyse bacterial cells]
Prepare Lysis Buffer + TCEP (final volume 160mL):
The recipe for Lysis Buffer per 20mL of volume is: 1 Roche cOmplete protease inhibitor tablet2uL DNase (RNase free)0.5mM PMSF final concentration
?. [Day 3: Lyse bacterial cells]
Add 8 Roche cOmplete protease inhibitor tablets.
?. [Day 3: Lyse bacterial cells]
Add  DNase (RNase free).
16 µl
?. [Day 3: Lyse bacterial cells]
Add PMSF to a final concentration of  ( of  stock PMSF).
800 µl
?. [Day 3: Lyse bacterial cells]
Add TCEP to a final concentration of  ( of  stock TCEP).
80 µl
?. [Day 4: SP Column Ion Exchange]
Prepare IEX Buffer A, using MilliQ water as solvent:
?. [Day 4: SP Column Ion Exchange]
Add  HEPES ().
?. [Day 4: SP Column Ion Exchange]
Add  NaCl.
?. [Day 4: SP Column Ion Exchange]
Add  glycerol.
?. [Day 4: SP Column Ion Exchange]
Add TCEP to final concentration .
?. [Day 4: SP Column Ion Exchange]
Prepare IEX Buffer B, using MilliQ water as solvent:
?. [Day 4: SP Column Ion Exchange]
Add  HEPES ().
?. [Day 4: SP Column Ion Exchange]
Add  NaCl.
?. [Day 4: SP Column Ion Exchange]
Add  glycerol.
?. [Day 4: SP Column Ion Exchange]
Add TCEP to final concentration .
?. [Day 4: SP Column Ion Exchange]
Run Äkta at 0.25 ml/min to attach column.
Use a 5 ml SP Column, Screw the bottom of column into top Äkta port, making sure to use wet connections. Use pre-column pressure alarm set at 0.5 MPa.
?. [Day 3: Lyse bacterial cells]
Fill to final volume of 160mL using MilliQ water.