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1 GENETIC ENGINEERING EXPERIMENT (Lab experiment book)
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Page 1: GENETIC ENGINEERING EXPERIMENT (Lab experiment book)structure2.konkuk.ac.kr/202001_GEE_lab_book_final.pdf · 2020-03-13 · GENETIC ENGINEERING EXPERIMENT (Lab experiment book) 2

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GENETIC ENGINEERING EXPERIMENT

(Lab experiment book)

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Week 3(3/16~20): General instructions for lab environments

Brief introductions for lab environments and works will be provided. Life as a research scientist

and basics of lab equipment are also discussed.

Following materials will be prepared by TAs for later experiments.

LB Media preparation protocol

❖ Introduction

Note: This protocol makes 500mL of broth or ~25 plates. In order for bacteria to be successfully

cultured, they must be grown in the appropriate media. LB, also known as Lysogeny broth, is a

nutrient rich broth that is a standard for culturing Escherichia coli, as it allows for quick growth

and high yields. Therefore, the proper preparation of LB will be crucial to maintaining our

bacterial stock throughout the summer. Furthermore, addition of agar to LB broth creates a gel

for bacteria to grow upon and is therefore used for plating bacterial cultures on petri dishes.

❖ Materials Reagents

● 5g Bacto-tryptone

● 2.5g yeast extract

● 5g NaCl

● 7.5g agar (Only necessary if making LB agar plates)

● 500mL of dH2O (distilled water)

❖ Procedure

➢ Part 1: Making the LB broth.

1. Obtain a clean 1L pyrex bottle

2. Obtain a graduated cylinder with 500mL of dH2O and add to the bottle. Record the amount

added.

3. Using filter paper, separately measure out 5g of NaCl, 5g of Tryptone, and 2.5g of yeast

extract on a scale and add them to the bottle. Swirl the bottle in a circular motion to mix.

Remember to re-calibrate your scales in between measurements.

4. If you are making LB agar plates, weigh and add 7.5g of agar and swirl to mix. Record the

amount added.

Note the contents do not necessarily need to be completely in solution before autoclaving.

➢ Part 2: Autoclaving

1. Lightly seal the top of the beaker with aluminium foil, and label the beaker with autoclave

tape stating LB (or LB agar)–[your name]–[date].

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2. Use appropriate transportation protocols to bring the LB bottle into the autoclave room.

Remember to store the beaker in an autoclavable basin, in case of spills.

3. Check the water level on the autoclave machine. Autoclave on the liquid setting for

approximately 20 min.

4. The contents of the beaker will be hot after autoclaving, therefore take the necessary measures

to prevent burns.

5. After autoclaving, allow the LB media to cool to 55°C before handling.

6. The LB broth can be stored in sterile conditions at room temperature and should be good for

3-4 months. Flame the lip of the bottle each time the LB is used. If the LB contains antibiotics,

store in a 4°C freezer.

● However, it is not recommended to store LB with antibiotics as the antibiotics will

degrade over time

➢ Part 3: Pouring the plates (for LB agar)

While pouring the plates, it is crucial to maintain a sterile environment.

1. Sterilize the workspace with 70% EtOH before depositing your materials. Light the Bunsen

burner.

2. Obtain a stack/roll of empty plates. The plates should still be in their plastic sleeve/wrapping,

as they should be sterile. It is essential that you minimize any chance of contaminating the plates.

Make sure that you open the package at the top and expose the plates as minimally as possible.

3. Once you take the plates out, store them upside down on your lab bench. Label the plates with

[your name]–[date]–[antibiotic].

4. Allow the LB media to cool before pouring. The LB will start to settle at ~30°C.

5. If you are preparing selective media, add antibiotic to the mixture. Swirl the flask in a circular

motion to mix. ● Use concentrated liquid stocks for the antibiotics.

6. Recommended antibiotic concentrations: ● Chloramphenicol (CAM): 25μg/mL ● Ampicillin

(AMP): 100μg/mL

7. Take an empty plate and open it slightly. You do not need to open it all the way to pour the

agar.

8. Pour agar until 2/3 of the plate has been covered, or approximately half of the plate has been

filled when viewed from the side. Pour the agar slowly to prevent the formation of bubbles. Swirl

the plate in a circular motion to distribute the media evenly on the plate.

10. After pouring, set the plates to cool in stacks of 4-5 to save space and flip the plates to

prevent condensation forming on the agar. Don’t stack plates too high - we want to minimize the

risk of spills. Allow the plates to cool for at least 20 minutes until the agar has solidified.

11. Rinse the Pyrex bottle with water before the remnants solidify and become hard to remove.

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12. The plates can then be stacked and stored in plastic bags (ideally, reuse the plastic bags that

the plates came in.)

13. Store LB agar plates in a 4°C freezer. They should be good for 1-2 months.

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Week 4(3/23~27): Buffer preparations (lysis buffer, purification buffer)

Almost all biochemical experiments are performed in solutions. How to prepare necessary

experimental solutions and reagents correctly is essential. We will discuss how to make solutions

accurately in lab.

Following buffers are examples of necessary solutions for protein purification.

Preparations of protein purification buffers

Materials

- 15 ml conical tube × 3

- Micropipette

- Stock solution

1M Tris-HCl pH 7.5, 5M NaCl, 1M Imidazole, 14.3M 2-mercaptoethanol, 100% Glycerol

- Distilled water

Method

1. Identification of the buffer working solution concentration.

The required buffer working solution concentration can be found in table(see below).

2. Calculate the volume of each stock solution for the working solution based on the table.

3. Add the calculated solution in a 15 ml conical tube using a micropipette.

4. Make up to 15ml with distilled water.

Lysis buffer Wash buffer Elution buffer

Stock solution Working solution

1M Tris-HCl pH 7.5 25mM 25mM 25mM

5M NaCl 300mM 1M 300mM

1M Imidazole 30mM 40mM 250mM

14.3M 2-

mercaptoethanol

0.5mM 0.5mM 0.5mM

100% Glycerol 10% 10% 10%

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Week. 5(3/30~4/3). Bacterial cell streaking/ cell stock, LB plate streaking

Creating bacterial stocks for long-term storage of plasmids

Protocol

1. Follow the steps for Inoculating an Overnight Liquid Culture.

2. After you have bacterial growth, add 500 μL of the overnight culture to 500μL of 50%

glycerol in a 2 mL screw top tube or cryovial and gently mix.

3. Freeze the glycerol stock tube at -80°C. The stock is now stable for years, as long as it is kept

at -80°C. Subsequent freeze and thaw cycles reduce shelf life.

4. To recover bacteria from your glycerol stock, open the tube and use a sterile loop, toothpick or

pipette tip to scrape some of the frozen bacteria off of the top. Do not let the glycerol stock

unthaw! Streak the bacteria onto an LB agar plate. Grow your bacteria overnight, and the next

day you will be able to start an overnight culture for plasmid DNA prep the following day.

Streaking and isolating bacteria on LB agar plate

Protocol

1. Obtain an LB agar plate with appropriate antibiotic.

2. Label the bottom of the plate with the plasmid name and the date. It is also a good idea to add

the antibiotic resistance and your initials.

3. Keep your lab bench area sterile by working near a flame or bunsen burner.

4. Using a sterile loop, pipette tip or toothpick, touch the bacteria growing within the punctured

area of the stab culture or the top of the glycerol stock.

5. Gently spread the bacteria over a section of the plate, as shown in the diagram to the right, to

create streak #1.

6. Using a fresh sterile pipette tip, toothpick, or freshly sterilized loop, drag through streak #1

and spread the bacteria over a second section of the plate, to create streak #2.

7. Using a third sterile pipette tip, toothpick, or sterilized loop, drag through streak #2 and spread

the bacteria over the last section of the plate, to create streak #3.

Streaking for a Single Colony

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8. Incubate plate with newly plated bacteria overnight (12-18 hours) at 37°C.

9. In the morning, single colonies should be visible. A single colony should look like a white dot

growing on the solid medium. This dot is composed of millions of genetically identical bacteria

that arose from a single bacterium. If the bacterial growth is too dense and you do not see single

colonies, re-streak onto a new agar plate to obtain single colonies.

10. Once you have single colonies, proceed to Recovering Plasmid DNA

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Week 6~8 (4/6~24): Protein Expression of Bacterial system

-From cell culture to cell collection-

Introduction

• pET system is for the cloning and expression of recombinant proteins in E. coli, Driven by

the strong bacteriophage T7 promoter and translation signals. The expression of T7

polymerase is controlled by Lac operon system. Expression of T7 polymerase is inactivated

by lac repressor without inducer. But when Isopropyl β-D-1-thiogalactopyranoside(IPTG),

a lactose analog, is present, then lac repressor is nullified and T7 polymerase is expressed.

This T7 polymerase binds exclusively to T7 promoter and translate protein gene it encodes.

T7 polymerase have very high level of translation rate compared to host polymerase and it

ensures high level of target protein expression. The pET System has continuously expanded

incorporating new technologies and options for expression. Collectively, this large

collection of pET vector types, different host strains and companion products ensures high

level of protein expression of various target protein sequence.

Method

⚫ Cell culture

1. Small scale culture

Pick a colony with 200ul micropipette tip. And inoculate 100ml LB media treated

with 100μl of ampicilin solution (100mg/ml). repeat ① to make 200ml of cell

media.

2. Inoculation

Inoculate each 2L LB media with 50ml of cell culture media from previous step.

Then treat 2L of LB media with 1ml of ampicilin.

3. Large scale culture

Culture E.coli cells until Optical density reaches 0.8 at 37°C.

⚫ Protein expression

1. Induction.

Then treat 2L LB media from previous step with 2ml of IPTG solution(1M).

2. Expression

Set the temperature of incubator 37°C. Incubate media for 8 hours

⚫ Cell collection

1. Centrifugation

Distribute 4L of LB media into 6 centrifuge bottles. Make sure each 2 facing

containers have same weight. Centrifuge at 4°C, 5000rpm for 30min. Repeat ①

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with same bottle to collect cell from 8L of media with least bottles. Make sure

sediments stacks on same side of bottle.

2. Freezing

Freeze cell sediments at -20°C refrigerator. Put bottles upside down to remove

remaining LB media.

Week 9: Mid-term exam

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Week 10(5/4~8): Sonication

Resuspend the cells in chilled lysis buffer. Normally ratios of cell wet weight to buffer volume of

1:1 to 1:4 are used.

Cool the cell suspension on ice for 10 min.

Sonicate the cell suspension with 10 short burst of 10 sec followed by intervals of 30 sec for

cooling.

⚫ Keep the suspension at all times on ice.

⚫ Avoid foaming.

⚫ Don’t go away while the sonicator is in operation. It is possible that the beaker breaks

or turns in the melting ice.

Remove cell debris by ultracentrifugation at 4°C for 1h at 13 000 rpm

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Week 11 (5/11~15): SDS PAGE-Preparation

An intact SDS PAGE electrophoresis system should include: a tank, lid with power cables,

electrode assembly, cell buffer dam, casting stands, casting frames, combs(usually 10-well or 15-

well), and glass plates (thickness 0.75mm or 1.0mm or 1.5mm). (Bio-rad brand one is

recommended)

The SDS PAGE gel in a single electrophoresis run can be divided into stacking gel and

separating gel. Stacking gel (acrylamide 5%) is poured on top of the separating gel (after

solidification) and a gel comb is inserted in the stacking gel. The acrylamide percentage in SDS

PAGE gel depends on the size of the target protein in the sample. (Details shown below)

Acrylamide % M.W. Range

7% 50 kDa - 500 kDa

10% 20 kDa - 300 kDa

12% 10 kDa - 200 kDa

15% 3 kDa - 100 kDa

• Volumes of stacking gel and separating gel differ according to the thickness of gel

casting:

Thickness of the gel Vol. of stacking gel Vol. of separating gel

0.75mm 2ml 4ml

1.0mm 3ml 6ml

1.5mm 4ml 8ml

• For a 5 ml stacking gel: (volume in ml)

H2O 2.975

0.5 M Tris-HCl, pH 6.8 1.25

10% (w/v) SDS 0.05

Acrylamide/Bis-acrylamide (30%/0.8% w/v) 0.67

10% (w/v) ammonium persulfate (AP) 0.05

TEMED 0.005

• For a 10ml separating gel: (volume in ml)

Acrylamide percentage 6% 8% 10% 12% 15%

H2O 5.2 4.6 3.8 3.2 2.2

1.5 M Tris-HCl, pH 8.8 2.6 2.6 2.6 2.6 2.6

10% (w/v) SDS 0.1 0.1 0.1 0.1 0.1

Acrylamide/Bis-acrylamide (30%/0.8% w/v) 2 2.6 3.4 4 5

10% (w/v) ammonium persulfate (AP) 0.1 0.1 0.1 0.1 0.1

TEMED 0.01 0.01 0.01 0.01 0.01

Note: AP and TEMED must be added right before each use.

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SDS PAGE making Protocol:

1. Clean the plates and combs.

For each gel, you will need one short plate, one spacer plate, and one comb. Spray a little bit of

70% ethanol on the plates, and wipe dry with Kimwipes. Wash the combs thoroughly with tap

water. It is critical to remove all dust and small particles, especially any bits of left-over

polyacrylamide.

1. Set-up the plates on the rack.

Layer the short plate on the spacer plate, with the spacers in between and slide the two plates into

the green holder. Make sure that the bottom edges of the two plates are flush to avoid leakage.

Lock the plates in, and place the holder on the rack, with the bottom edges of the plates pushed

into the gray foam gasket to make a water-tight seal. Test the seal by pipetting or squirting a

small volume of water between the plates and making sure there is no leakage. Blot dry with

filter paper.

2. Make the separating gel:

Set the casting frames (clamp two glass plates in the casting frames) on the casting stands.

Prepare the gel solution (as described above) in a separate small beaker.

Once TEMED is added, the gel will begin to polymerize, so you need to work fast (but

carefully).

Swirl the solution gently but thoroughly.

Pipette the gel mix into the gap between the glass plates, making sure you leave enough space at

the top for the stacking gel and comb.

To make the top of the separating gel be horizontal, fill in isopropanol into the gap until an

overflow.

Wait for 20-30min to let it gelate.

3. Make the stacking gel:

Discard the isopropanol and you can see separating gel left.

Pipet in stacking gel until an overflow.

Insert the well-forming comb without trapping air under the teeth. Wait for 20-30min to let it

gelate.

4. Make sure a complete gelation of the stacking gel and take out the comb.

Take the glass plates out of the casting frame and set them in the cell buffer dam. Pour the

running buffer (electrophoresis buffer) into the inner chamber and keep pouring after overflow

until the buffer surface reaches the required level in the outer chamber.

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Week 12 (5/18~22): protein affinity purification/ Ni column running/ SDS running sample

making

Protein affinity purification/ Ni column running

Materials

- 6ml column with Ni-NTA resin

- Bacterial cell lysate

- lysis buffer

- Washing buffer

- Elution buffer

- E-tubes to collect the proteins

Ni-NTA affinity purification method

Using the lysis buffers, columns and cell lysate, follow the procedure below to purify

proteins under native conditions:

1. Equilibrate the Ni-NTA resin with lysis buffer solution with 5x column volume(1ml).

2. Collect all solutions from column by 1ml in E-tube.

3. Add lysate prepared under native conditions to a prepared Purification Column.

4. Add wash buffer to column with 5x column volume (1ml).

5. Add elution solution to column with 5x column volume (1ml).

6. Add column storage solution (water) with 5x column volume.

SDS running sample making

Material

- 6×SDS sample loading buffer

60% (v/v) Glycerol, 12% SDS, 300 mM Tris-HCl pH 6.8, 30% b-mecaptoethanol, 0.05%

bromophenol Blue

- E-tubes

- Micropipette

- water bath

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Methods

1. Add one volume of SDS-PAGE Sample Loading Buffer [6X] to five volumes of protein

solution. For example add 5µl SDS-PAGE Sample Loading Buffer [6X] to 25µl protein solution.

2. Vortex the tube to mix the contents. Place the sample tube in a boiling water bath (95℃) for 5-

10 minutes.

4. After the boiling is complete, vortex and centrifuge the tube for 30 seconds. The sample is

now ready for loading on SDS-PAGE gels. Vortex the tube before loading the protein solution

on the gel.

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Week. 13(5/25~29): SDS page running/staining, destaining

Running the Gel

1. Remove comb from the acrylamide gel and assemble cast gel into running module.

2. Add freshly prepared 1x running buffer (300 ml) to both chambers of the apparatus.

1x SDS-PAGE Running Buffer (30.3 g Tris base, 144.0 g Glycine, 10.0 g SDS)

3. Load the prepared samples into the wells of the gel.

4. Run the gel at 180 V until the dye front reaches the bottom of the gel (~90 min).

Staining & Destaining the Gel

1. Remove the run gel from the aparatus and remove the spacers and glass plates. Place the gel

into a small tray. Note: Never us a metal spatula to separate the glass plates.

2. Add staining solution and stain for > 30 min with gentle shaking.

*Coomassie Stain Solution (Ethanol 150 ml, Glacial Acetic Acid 50 ml DDI H2O 300 ml)

3. Pour off and save the stain.

4. Add destain solution and destain for ~1 min with gentle shaking.

*Destain Solution (Ethanol 1200 ml, Glacial Acetic Acid 400 ml DDI H2O 2.4 L)

5. Pour off and discard the destain solution. Add ~ 30 ml of destain solution.

6. Destain with gentle shaking until the gel is visibly destained (> 2 hr).

7. Pour off and discard the destain solution.

8. Rinse with DDI H2O. Add DDI H2O and rinse with gentle shaking.

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Week 14(6/1~5):

Protein concentration measurement

Introduction

• There are several different ways to measure protein concentration. But in this experiment we

will use Bradford assay method for it’s convenience and wide prevalence in lab protocols.

Bradford assay use dye-based protein assay method. Bradford solution contains ‘Coomassie

brilliant blue G 250’ dye. It can exist in three forms. Anionic, cationic and neutral form. In protein-

free solution, dye prefers to be exists in cationic form, which absorbs light around 470nm

wavelength(red). But when protein molecule is added to this solution, noncovalent interaction

between dye and peptide backbone stabilize the anionic form, which absorbs light of wavelength

595nm(blue). Hence we can measure the concentration of protein in solution with spectrometer.

• Spectrometer uses Beer lambert law to measure the absorbance of sample. Beer lambert law is

equation showing linear relationship between absorbance and concentration of an absorbing

species. Beer lambert law is usually shown as below.

A = a( ) * b * c

○a ( ) is a wavelength-dependent absorptivity coefficient. ○b is the path length, ○c is the

concentration of analyte(sample). When this equation is combined with equation explaining

Transmittance ( T=I/I0 ) and equation exchanging transmittance into absorbance ( A=-log T ), we

can measure the concentration of sample.

Method

1. Add 5μl, 10μl, 15μl, 20μl and 25μl of BSA solution(1mg/ml) to 1ml Bradford solution in

cuvette and measure OD595.

2. Add 10μl of sample X to Bradford solution and measure OD595.

3. Calculate the protein concentration of sample X with linear regression equation acquired

from ①.

4. Add 40μl and 50μl of BSA solution to Bradford solution and measure OD595 value.

5. Compare the actual OD595value and theoretical value from OD595linear regression equation.

Question

1. how was the protein concentration of sample X?

2. Were the theoretical value and actual value of 40μl and 50μl of BSA solution match well? If

not, why do u think these values doesn’t match well?

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3. What should we do to avoid problem of ○2 ?

Dialysis

Introduction

➢ Dialysis is a step to remove eluent and salt from purified protein solution and preparing for next

step of purification. Dialysis uses semipermeable membrane to diffuse salt and eluent while

maintaining protein concentration. Semipermeable membrane has small pores which is big

enough for salt ions and eluents to pass through, but narrow enough to keep proteins inside.

Method

1. Prepare 3L of dialysis buffer. (Tris-HCl pH7.5 25mM, glycerol 10%) put it into 4°C refrigerator.

2. Pull out 15cm of semipermeable tube. Soak it with dialysis buffer and tightly clamp one side of

tube.

3. Measure the concentration of protein solution with spectrometer.

4. Pour in 10ml of protein solution containing 1M of Ni2+ ions. (Ni2+ ions are just for visualization of

salts. It won’t be used in real experiment protocols.)

5. Clamp the remaining side of tube and insert it into styrofoam buoy and put it into dialysis buffer.

6. Put stirring bar into dialysis buffer and stir it for 16hours in 4°C refrigerator.

7. Pour out protein solution from semipermeable membrane and check color and protein

concentration of solution.

Question

1. How was the color and protein concentration of the protein solution after dialysis and what does

it mean?

2. If the initial NaCl concentration was 1M, then what would be theoretical concentration of slat

after dialysis?

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Week 15 (6/8~12): Ion exchange column chromatography

1. Buffer preparation

Buffer pH and ionic strength are crucial for all forms of ion exchange chromatography. It is

best to readjust buffer pH after adjusting salt concentration and ensure that buffer counterions

are compatible. Buffer counterions should have the same charge as the resin; for positively

charged anion exchange resins, Tris buffers are an excellent choice.

2. Column equilibration

Equilibrate the column until pH and conductivity readings stabilize (typically requires ≥5

column volumes of buffer).

3. Sample loading

Because ionic strength and pH are main determinants of protein binding to IEX resins,

whenever possible, load the sample in the starting buffer.

4. Column washing

Wash the column in loading buffer (0% Buffer B) until no protein is detected in the

flowthrough (3–5 column volumes).

5. Elution

Protein can be eluted either by a linear gradient elution or using a step isocratic elution. Often,

a gradient elution may be used to optimize elution conditions. Once the elution profile of the

protein of interest has been established and it is known at what ionic strength or pH a protein

elutes, a step elution can be used to speed the purification process.

Week 16 (6/15~19): Lab summary


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