Difference between revisions of "Team:UC Davis/Safety"

Preparation of DNA for Cloning
Originally, our cloning method was to be Gibson assembly. We ordered our 8 genes with 5’ and 3’ gibson overhangs from IDT. Once received, we rehydrated the genes:

1. Centrifuge for 3-5 seconds at >3000 x g to pellet at the bottom
2. Add 20 uL TE buffer or Milli-Q water for a final concentration of 10ng/uL
3. Briefly vortex and centrifuge
4. Store at -20 degrees for up to 24 months with TE buffer or 1 month with dH2O

We made four 50 mL and four 2 mL overnight cultures of DH10B containing pET29b+ vector for insertion with kanamycin resistance selection. The grown 50 mL cultures were then transferred to 50 mL falcon tubes and spun down to pellet at 4000 x g for 10 minutes. We then followed the protocol included in the Invitrogen MaxiPrep kit used. The results gave an average of 1.93 ng/uL, which is below the threshold for accurate concentration measurement for the Biotek Epoch spectrophotometer with the Take3 Microvolume plate. This also showed that the results yielded next to no DNA for the MaxiPrep and decided to go forth with a MiniPrep on the four 2 mL cultures using the Invitrogen MiniPrep protocol provided with the kit. The results yielded an average of 90 ng/uL which is above the 60-80 ng/uL concentration needed for the restriction digestion.

Double digestion:

We used the NEB double digest finder to select which buffer and protocol to use for our reaction. Since we used NdeI and EcoRI as our restriction sites, the highest activity without having star activity was NEBuffer 3.1 with 100% activity in NdeI and 50% activity in EcoRI. 50 uL double digest reactions were made of the miniprep, containing 1 uL of each restriction enzyme, 5 uL of the 10x 3.1 NEBuffer, and the remaining 43 uL of our miniprepped plasmid and allowed to incubate at 37 degrees for one hour.

Gel separation and purification:

Each 50 uL sample received 10 uL of NEB 6x loading dye and was run on a 1% agarose gel alongside uncut plasmid and O’Generuler 1kb Plus (Thermofisher). The gel was analysed on a [name of gel machine] and the lines of linear plasmid were cut out and placed in premeasured 2 mL eppendorf micro centrifuge tubes and weighed again. Gel purification was completed with the QIAprep Gel Extraction Kit, following the protocol provided with the kit. The average concentration of the DNA from the gel purification was about 14 ng/uL, much less than wanted for the next step.

Gibson assembly:

5 uL gibson reaction were performed with 2.5 uL of gibson master mix and with rest having equimolar amounts of linear vector and gene. The reactions were placed in a 50 degree dry bath and left to react for 1 hour.

Transformation:

The 5 uL of DNA from the Gibson reactions were then loaded onto Millipore (type VSWP) drop dialysis film with 0.025 μm on ~40 uL of ultra-pure Milli-Q water for desalting over the course of one hour. 2 uL of dialyzed DNA along with 30 uL of electrocompetent BLR cells were transferred to Bio Rad 0.1 cm gap Gene Pulser cuvettes for electroporation and kept chilled. Electroporation was conducted with a Bio-Rad MicroPulser Electroporator set to pulse on the bacteria setting. After electroporation, the cells were immediately transferred to 200 L of TB and placed in a 37° incubator to recover for an hour. The cells were then added to LB agar plates with kanamycin and spread with ~10 sterile glass beads per plate. The beads were then dumped into 70% ethanol and the plates were allowed to dry in the incubator at 37 degrees for 30 minutes with the lid side up. The plates were then flipped lid side down and incubated at 37 degrees overnight.

CPEC reaction:

1. Each reaction contained:
&nbsp&nbsp&nbsp&nbsp&nbsp&nbspa. 0.5 uL of Phusion High-Fidelity DNA Polymerase (Thermofisher) - added last
&nbsp&nbsp&nbsp&nbsp&nbsp&nbspb. 10 uL of 5x Phusion reaction buffer
&nbsp&nbsp&nbsp&nbsp&nbsp&nbspc. 2.5 uL of forward oligo
&nbsp&nbsp&nbsp&nbsp&nbsp&nbspd. 2.5 uL of reverse oligo
&nbsp&nbsp&nbsp&nbsp&nbsp&nbspe. 1 uL of 10 uM dNTPs
&nbsp&nbsp&nbsp&nbsp&nbsp&nbspf. 1 ng of template DNA
2. Thermocycler settings:
&nbsp&nbsp&nbsp&nbsp&nbsp&nbsp- 30 seconds at 98 degrees - 1 cycle
&nbsp&nbsp&nbsp&nbsp&nbsp&nbsp- 10 seconds at 98 degrees \
&nbsp&nbsp&nbsp&nbsp&nbsp&nbsp- 30 seconds at 55 degrees # of cylces should exceed # of assembly pieces
&nbsp&nbsp&nbsp&nbsp&nbsp&nbsp- 15 seconds per kb at 72 degrees/
&nbsp&nbsp&nbsp&nbsp&nbsp&nbsp- 10 minutes at 72 degrees - 1 cycle
The PCR products of both the insert and the linearized plasmid were then combined in equimolar amounts and a CPEC was done on the combination.

Transformation:

5 uL of each of the CPEC reactions were then loaded onto Millipore (type VSWP) drop dialysis film with 0.025 μm on ~40 uL of ultra-pure Milli-Q water for desalting over the course of one hour. 2 uL of dialyzed DNA along with 30 uL of electrocompetent DH10B cells for half and BLR cells for the other half were transferred to Bio Rad 0.1 cm gap Gene Pulser cuvettes for electroporation and kept chilled. Electroporation was conducted with a Bio-Rad MicroPulser Electroporator set to pulse on the bacteria setting. After electroporation, the cells were immediately transferred to 200 L of TB and placed in a 37° incubator to recover for an hour. The cells were then added to LB agar plates with kanamycin and spread with ~10 sterile glass beads per plate. The beads were then dumped into 70% ethanol and the plates were allowed to dry in the incubator at 37 degrees for 30 minutes with the lid side up. The plates were then flipped lid side down and incubated at 37 degrees overnight.

Checking for colonies and sequencing:

The resulting plates yielded very few colonies (1-3) for the BLR strains that did have colonies and the DH10B strains with too many to count. A colony from each plate was selected and grown overnight in 2 mL culture tubes with kanamycin. The cultures were then miniprepped with the QIAprep Spin Miniprep Kit and sent to Eurofins for sequencing. The colonies from the BLR strains showed recircularized plasmid while the DH10B strains showed proper gene insertion.

Transformation for Expression:

The leftover miniprepped plasmid that was sent for sequencing and confirmed to contain the genes of choice were then loaded onto Millipore (type VSWP) drop dialysis film with 0.025 μm on ~40 uL of ultra-pure Milli-Q water for desalting over the course of one hour. 2 uL of dialyzed DNA along with 30 uL of electrocompetent BLR cells were transferred to Bio Rad 0.1 cm gap Gene Pulser cuvettes for electroporation and kept chilled. Electroporation was conducted with a Bio-Rad MicroPulser Electroporator set to pulse on the bacteria setting. After electroporation, the cells were immediately transferred to 200 L of TB and placed in a 37° incubator to recover for an hour. The cells were then added to LB agar plates with kanamycin and spread with ~10 sterile glass beads per plate. The beads were then dumped into 70% ethanol and the plates were allowed to dry in the incubator at 37 degrees for 30 minutes with the lid side up. The plates were then flipped lid side down and incubated at 37 degrees overnight.

Checking for colonies and glycerol stock for culture growth:

Colonies were seen on all of the plates and contained too many to count. Using a pipet tip, one colony from each of the plates were selected and mixed into 2 mL of TB with kanamycin for overnight growth. The cells, after ~16 hours had 2 mL of 50% glycerol stock solution added to achieve 4 mL total and were aliquoted out into 1.5 mL eppendorf microfuge tubes containing 1 mL of 25% glycerol cultures. These glycerol stock cells were kept in a -80 degree freezer until needed for expression growth cultures.

Cell growth for expression:

Using 50 mL falcon tubes, 25 mL of TB containing kanamycin were poured into each tube. Using the glycerol stock cells on ice, a pipet tip was used to obtain a fleck of frozen cultures and carefully mixed for each of the designated tubes. The cultures were shaken for ~24 hours at 300 rpm in 37 degrees.

Expression and Protein Purification:

Autoinduction media for n + 1 samples
25 mL * (n + 1) samples
&nbsp&nbsp&nbsp&nbsp&nbsp&nbsp1000x Trace metals mix
&nbsp&nbsp&nbsp&nbsp&nbsp&nbsp1000x Kanamycin
&nbsp&nbsp&nbsp&nbsp&nbsp&nbsp1000x MgSO4
&nbsp&nbsp&nbsp&nbsp&nbsp&nbsp50x 50:52
&nbsp&nbsp&nbsp&nbsp&nbsp&nbsp20x NPS
&nbsp&nbsp&nbsp&nbsp&nbsp&nbspTB to reach final volume
To express protein: &nbsp&nbsp&nbsp&nbsp&nbsp&nbspA colony was grown overnight in 25 mL TB containing kanamycin in 50 mL falcon tubes at 37C at 300 rpm. The resulting cell cultures were then spun down in a centrifuge at 4700 rpm for 10 min, supernatant was removed, and the pellets were resuspended in 25 mL autoinduction media. These cultures were then allowed to express for 24-36 hours at 18 degrees at 300 rpm. Cells were spun down and supernatant was removed. Cells were stored in -20 until the protein was needed. Protein was always purified the same day they were assayed to assure quality. Protein Purification: -Dispense 500 uL aliquots of lysis solution to 2 mL microcentrifuge tubes
- Resuspend cells in 500 uL wash buffer and vortex
- Transfer 1 mL resuspension to tubes with lysis solution
- Seal put on rocker for 20 minutes
- Spin in micro centrifuge at 14,700 rpm for 20 minutes
- While spinning, prepare micro columns in rack over waste collector
- Add 100 uL Co2+ or Ni2+ to each column
- Add 1 column volume (1 mL) wash buffer
- Once 20 minute spin is complete, load clarified supernatant to column
- Wash 6 times with 500 uL wash buffer
- Transfer columns to fresh 2 mL microcentrifuge tubes
- Elute with 100 uL elution buffer, wait 2-3 min
- Transfer columns to fresh 2 mL microcentrifuge tubes
- Elute with 100 uL elution buffer, wait 2-3 min
- Transfer columns to fresh 2 mL microcentrifuge tubes
- Elute with 100 uL elution buffer, wait 2-3 min
- Briefly spin so all protein elutes in final fraction
- Check concentration of each elution from A280
- Check purity from SDS-PAGE gel

For SDS-PAGE gel:
4x denaturing buffer: Add 100 uL beta-mercapto ethanol to 900 uL Laemmli sample bufer in fume hood.

Mix 12 uL of protein with 4 uL denaturing buffer in PCR tubes and heat in thermocycler at 100C for 10 min. Load 12 uL of denatured protein onto gels. Run at 200 V for 20 min.

Enzyme activity assay with natural substrate analog crotonyl CoA:

in 96 well plate:

Each well had 50 uL enzyme
25 uL 100 uM NADH (final concentration)
25 uL 100 uM crotonyl CoA (final concentration)

Negative control:
25 uL 100 uM NADH (final concentration)
25 uL 100 uM crotonyl CoA (final concentration)
50 uL protein buffer

Reactions were measured spectrophotometrically in either a Biotek plate reader or manually in cuvettes in the Io Rodeo colorimeter by measuring the decrease in NADH at 340 and/or 365 nm. Reading were taken every minute over an hour.

To find the minimum amount of enzyme needed to still see enzyme activity, each enzyme was diluted 10 fold up to a million fold in PCR tubes. Assay was run same as just described.

Enzyme activity assay with alternative substrates:

5 mM substrates used on max concentration of enzyme that elutes
500 uM NADH


To measure enzyme inhibition by triclosan:

Assayed contained 10 uM, 50 nM, and 1 nM triclosan with ~2 nM Enzyme diluted approximately 10,000x. 100 uM NADH, 100 uM Crotonyl CoA

NADH oxidation was measured spectrophotometrically at eithe 340 nm or 365 nm every minute over the course of an hour

Buffers:

Lysis Solution:

250 uL Bugbuster * (n+1) samples
350 uL wash buffer *(n+1) samples
1 mg dry lysis mix * (n+1) samples
PBS: 10x (pH 7.4)(1L)
-80g Nacl
-2.0g KCl
-14.4g Na2HPO4
-2.4g KH2PO4
*adjust pH to 7.4

Wash Buffer: (1x PBS, pH 7.4, 10mM Imidazole)(2L)
-200mL 10x PBS
-20mL Imidazole
-Milli-Q water up to 2L
*adjust pH if necessary

Elution Buffer: (pH 7.5)(1L)
-11.9g HEPES
-8.7g NaCl
-7.31g EDTA
-Milli-Q water up to 1L
*adjust pH to 7.50

Wash Buffer (1X PBS, pH 7.4, 10mM Imidazole) (2L)
200mL 10X PBS
20mL 1M imidazole
*bring up to 2L with ddH20 and adjust pH if necessary

Elution Buffer (1X PBS, pH 7.4, 200mM Imidazole) (1L)
100mL 10X PBS
200mL 1M imidazole
*bring up to 1L with ddH20 and adjust pH if necessary

20X NPS – 1L
Water 900mL
(NH4)2SO4 66g
KH2PO4 (monobasic) 136g
Na2HPO4 142g

1000x Trace Metals Mix - 100ml
Compound Amount
Water 36ml
0.1M FeCl3-6H2O 50ml
1M CaCl2 2ml
1M MnCl2-4H2O 1ml
1M ZnSO4-7H2O 1ml
0.2M CoCl2-6H2O 1ml
0.1M CuCl2-H2O 2ml
0.2M NiCl2-6H2O 1ml
0.1M Na2MoO4-2H2O 2ml
0.1M Na2SeO3-5H20 2ml
0.1M H3BO3 2ml
*SLOWLY ADD SOLIDS WHILE STIRRING, After dissolved sterile filter!

1M MgSO4 - 300ml
Compound Amount
water 300ml
MgSO4 74g
After dissolved, sterile filter

50x 5052 - 1L
5052 = 0.5% glycerol, 0.05% glucose, 0.2% alpha-lactose
Compound Amount
water 730ml
Glycerol 250g
Glucose 25g
a- lactose (D+) 100g
Can speed up dissolving by heating in the microwave… don’t get too hot or boil…
Sterile Filter
Store in refrigerator.