iGEM 2023 — Team plklfc-1


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# Engineering Success

**Cycle 1:**

**Design**

Our antibodies are composed of inserts including a ribosomal binding site (RBS), the antibody coding sequence, and a terminator. Non-illegal restriction sites were also placed in the middle of each component mentioned above. Initially we have decided to use a low-copy plasmid of pSB3K3 for our vector due to its ability to lower bacterial burden so as to uplift the growth rate of the strain. This is a crucial factor for us to obtain pure antibodies and hamper over-expression of antibodies instead of antibiotic resistance genes.

**Build**

We carried out restricted digestion to generate compatible end to ligase phosphodiester bond our insert into the plasmid pSB3K3 and upon transformation to transfer the recombinant plasmid to the vector bacteria and grow bacteria on agar plate with kanamycin.

**Test**

Nevertheless, experimental results unveiled that the expression level was extremely low. The transformation efficiency of pSB3K3 was also poor as shown from the data. There was miniscule growth of bacteria with the plasmid inserted as no colonels were seen on the plate.

**Learn**

We discerned that as pSB3K3 has kanamycin resistance, the selection of bacteria containing pSB3K3 has to use kanamycin but at the same time it will inhibit protein expression level. We then further delved into the issue by in-depth research and we came across the 2022 Virginia iGEM team that confronted the same challenge and obstacle as us. Based on the experimental results from the team and other scientific journals, they proved our previous direction to be mistaken as we should opt for vectors with a higher expression level instead of pSB3K3.

**Cycle 2:**

**Design**

We have now opted for the pET-21b expression vector which is a high-copy number plasmid and the reason for such selection is its high expression level. The selection of bacteria containing pET-21b requires the antibiotic ampicillin, it has ampicillin resistance which will not be killed by this antibiotic, and most importantly ampicillin will not inhibit protein expression level like kanamycin.

**Learn**

We succeeded in binding the antibody with antigen after using pET-21b. Click the results page to check out our results. For future improvement, the concentration of our antibody checked according to OD is slightly low so we should use another plasmid or modify pET-21b to higher the concentration of antibodies obtained.

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# Polymerase Chain Reaction (PCR)

---

![](https://static.igem.wiki/teams/4923/wiki/experiment7.png)

---

**Experiment Description:**

Using the ability of DNA polymerase to synthesise new strands of DNA complementary to allow rapid amplification of a specific segment of DNA.

Chemicals required:

●
2X master mix (contain MgCl2, dNTPS, DNA polymerase)

●
Distilled water

●
DNA sample

●
Primer

●
TE buffer

Instruments required:

●
Ice bucket

●
Pipette

●
Pipette tip

●
PCR tube

Apparatus required:

●
Mini Centrifµge

●
Thermal cycler

●
Water bath

**Before starting:**

1. Resuspending
   Gene Fragments (1000 ng after delivery)

2. Centrifµge the tube at 3000 ×g
   to ensure that the material is at the bottom of the tube.

3. Add
   100µl TE buffer to reach a final concentration of 10ng/µl.

4. Vortex
   briefly.

5. Incubate
   at 50°C for 20 minutes.

6. Briefly
   vortex and centrifµge the tube.

(Storage: store
the DNA at –20°C)

Resuspending of dry oligo (primer) (2 nmol
after delivery)

1. Centrifµge the tubes before
   opening them.

2. Add a
   200µl TE buffer to reach a final concentration of 10μM.

3. Store
   the standard DNA oligonucleotide at –20°C.

**PCR Protocol step:**

1. Keep
   all components on the ice.

2. Mix the
   following solution in per tube (repeat six times using different amount of
   DNA):

●
2µl primer

●
25µl 2x master mix

●
Template dna
(0.5µl / 1µl /2µl/ 3µl/ 4µl/ 5µl)

●
Distilled
water(22.5µl/22µl/21µl/20µl/19µl/18µl)

3. Vortex
   and centrifµge the mixture for 30 seconds and 1
   min respectively.

4. Put the
   sample to the thermal cycler.

5. Start
   the thermal cycler with the following setting:

●
Initial denaturing: 95°C for 5
mins

●
Denaturing: 94°C for 30s

●
Annealing: 60°C for 40s

●
Extending: 72°C for 40s

●
Repeat 5.2-5.4 for 20-40 times

●
Final extension: 72°C for 5
mins

●
Holding step in 4°C for short
time storage

# PCR Purification

---

**Experiment Description:**

Remove
enzymes, nucleotides, primers and buffer components from PCR products in order to
obtain a purified gene segment.

Chemicals required:

●
Monarch® PCR & DNA Cleanup Kit:

●
DNA Wash Buffer (add 100 ml of
ethanol to the Monarch DNA Wash Buffer)

●
DNA Cleanup
Binding Buffer (add 63.5 ml of isopropanol to the DNA Cleanup
Binding Buffer.)

●
DNA Elution Buffer

Instruments required:

●
Pipette

●
Pipette tip

●
PCR tube

●
DNA Cleanup
Columns (5 μg)

●
Collection Tubes (2 ml)

●
1.5 ml microfµge
tube

Apparatus required:

●
Centrifµge

**Before starting:**

1. Add 100 ml of ≥ 95% ethanol
   to 25 ml of DNA Wash Buffer.

2. (Optional)
   Add 0.36 volumes of isopropanol to one volume of DNA Cleanup
   Binding Buffer.

**All centrifµgation steps shoµld be
carried out at 16,000 x g (~13K RPM in a typical microcentrifµge)**

**PCR Purification Protocol step:**

1. Pipette
   25µl of sample to another PCR tube (total two solutions).

2. Dilute
   sample with 12.5µl DNA Cleanup Binding Buffer.

3. Mix
   well by pipetting up and down or flicking the tube. Do not vortex.

4. Insert
   column into collection tube and load sample onto column and close the cap.
   Spin for 1 minute, then discard flow-throµgh.

5. Re-insert
   column into collection tube. Add 200 μl DNA
   Wash Buffer and spin for 1 minute. Discarding flow-throµgh.

6. Repeat
   wash (Step 5)

7. Transfer
   column to a clean 1.5 ml microfµge tube. Use
   care to ensure that the tip of the column does not come into contact with
   the flow-throµgh.

8. Add 20 μl of DNA Elution Buffer to the centre of the
   matrix. Wait for 1 minute, then spin for 1 minute.

9. Collect
   the eluted DNA in a microcentrifµge tube.

10. Repeat
    2-9 with another PCR tube containing PCR product.

# Agarose Gel Electrophoresis (PCR)

---

![](https://static.igem.wiki/teams/4923/wiki/experiment27.png)

---

**Agarose Gel Electrophoresis**

**Experiment description:**

Separating DNA fragments based on their size and charge to
determine the size of gene by comparing the bands of the DNA samples with DNA
ladder.

Chemicals required:

●
1x TAE 40ml

●
Agarose powder

●
Ethidium bromide/dna stain

●
6X loading buffer

●
1Kb DNA ladder

Instruments required:

●
Microwavable bottle

●
Spatµla

●
Micropipette

●
Micropipette tip

●
Casting tray

●
Well Combs

●
Gel box

Apparatus required:

●
Electrical balance

●
Microwave

●
Voltage source

●
UV light source

**Gel Electrophoresis Protocol step:**

**Preparation of gel:**

1. Measure
   0.36 g of agarose.

2. Mix
   agarose powder with 30 mL 1xTAE in a microwavable flask.

3. Microwave
   the bottle with the lid not fµlly covered until
   bubbles evolve from the solution can be seen(1-2mins), Remove with gloves
   and swirl.

4. Swirl
   around to ensure the dissolving of the powder to confirm there are no
   white precipitates in the liquid.

5. If
   precipitation is still present, repeat step 3-4.

6. Cool
   down the solution by placing the bottle under running water for 1 mins

(Let agarose solution
cool down to about when you can comfortably keep your hand on the flask for
10s)

7. Add 1 μL of ethidium bromide or DNA staining to
   solution and swirl.

8. Pour
   the solution into the casting tray. Insert the comb into the top of the
   gel and allow the gel to solidify for 30 min. Avoid bubbles in the gel.

9. Rinse
   with water and dry the flask to prevent residual gel from solidifying in
   the flask.

**Running Gel Electrophoresis:**

1. Once
   the gel has solidified, carefµlly remove the
   comb by pµlling straight up.

2. Prepare
   the DNA samples by adding a 6X loading buffer to each.

(If doing a gel
extraction in an 8-well gel, combine 30 μL DNA
with 6 μL 6X loading buffer to load 36 μL.)

3. Fill
   the gel box with 1xTAE (or TBE) until the gel is covered.

4. Load 5 μL of DNA ladder into one lane of your gel.

5. Load
   samples into wells. Avoid bubbles.

6. Place
   the lid on the casting tray and ensure the red and black wires are
   connected to the matching red and black electrodes on the casting tray.

7. Run gel
   at 90V for 30-50 min.

8. Turn
   OFF power, disconnect the electrodes from the power source, and then carefµlly remove the gel from the gel box.

**Gel Extraction and Purification**

**Experiment description:**

Isolating DNA from an agarose gel to obtain a purified dna product from agarose gel.

Chemicals required:

●
Ethanol 96-100%.

●
3M sodium acetate, pH 5.2 (may
be needed).

●
GeneJET Gel Extraction Kit

●
Binding Buffer

●
Wash Buffer
(concentrated)(45ml)

●
Elution Buffer

●
GeneJET Purification Columns

Instruments required:

●
Sterile razor blade

●
Tweezers

●
Pipette

●
Pipette tip

●
1.5ml microcentrifµge
tubes

Apparatus required:

●
Gel imager

●
Microcentrifµge

●
Water bath

●
Vortex generator

**Before starting:**

1. Dilute
   the Wash Buffer (concentrated) with 225 mL ethanol

2. Examine
   the Binding Buffer for precipitates before each use. Re-dissolve any
   precipitate by warming the solution to 37 °C and cooling to 25 °C.

(Note:All centrifµgations
shoµld be carried out in a table-top microcentrifµge at >12000 rpm (10 000-14 000 rpm))

**Gel Extraction and Purification
Protocol step:**

1. Once
   you have run your gel, move it to an open UV box, remove it from any gel
   tray.

2. Excise
   gel slice containing the DNA fragment using a clean scalpel. Cut as close
   to the DNA as possible to minimise the gel volume.

3. With
   sterile tweezers, Place the gel slice into a pre-weighed 1.5 mL tube and
   weigh. Record the weight of the gel slice.

4. Add 1:1
   volume of Binding Buffer to the gel slice

(volume: weight)
(e.g add 100 μL of
Binding Buffer for every 100 mg of agarose gel).

5. Incubate
   the gel mixture at 50-60 °C for 10 min or until the gel slice is
   completely dissolved. Mix the tube by inversion every few minutes to
   facilitate the melting process.

6. Vortex
   the gel mixture briefly to ensure that the gel is completely dissolved.

7. Check
   the colour of the solution. A yellow colour indicates an optimal pH for
   DNA binding. If the colour of the solution is orange or violet, add 10 μL of 3 M sodium acetate, pH 5.2 solution and
   mix. The colour of the mix will become yellow.

8. Transfer
   up to 800 μL of the solubilized gel
   solution (from step 3 or 4) to the purification column. Centrifµge for 1 min. Discard the flow-throµgh
   and place the column back into the same collection tube.

9. Add 700
   μL of Wash Buffer to the GeneJET purification column. Centrifµge
   for 1 min. Discard the flow-throµgh and place
   the column back into the same collection tube.

10. Centrifµge the GeneJET purification column for an additional 1 min to
    completely remove residual wash buffer.

11. Transfer
    the GeneJET purification column into a clean 1.5
    mL microcentrifµge tube. Add 50 μL of Elution Buffer to the centre of the
    purification column membrane. Centrifµge for 1
    min.

12. Discard
    the GeneJET purification column and store the
    purified DNA at -20 °C.

# Plasmid Extraction

---

![](https://static.igem.wiki/teams/4923/wiki/experiment27.png)

---

**Experiment description:**

Throµgh a series of experiments, extract the plasmids inside the
bacteria.

Experimental flow:

1. Preparation of agar plate

2. Isolating Bacteria on an
LB Agar Plate

3. Inocµlating a
Liquid Bacterial Cµlture

4. Plasmid extraction

Or

Creating Bacterial Glycerol Stocks (for Long-term Storage of Plasmids)

**1. Preparation of Agar Plate**

Chemicals required:

●
Pre-mixed agar powder

●
10 mg/mL ampicillin (from
stock)

●
Sterile H2O

Instruments required :

●
Autoclavable flasks

●
Ice bucket to hold antibiotic

●
Sterile plates

●
Marker

Apparatus required:

●
Autoclave

●
Water bath

**Preparation of Agar Plate Protocol
step:**

1. Measure
   37g of pre-mixed LB-agar powder per L of molten agar you’d like to make.
   (The precise mass you measure out will be based on the number of plates
   you’d like to pour).

2. Transfer
   the LB-agar powder you’ve measured out into an appropriately sized bottle
   for autoclaving.

3. Transfer
   the sterile water (in our case 500 ml) to the same bottle and swirl to
   form a medium.

4. Cover
   the opening of the bottle with its cap or aluminium foil (but do not make
   an air- tight seal!) and tape the bottle with autoclave tape.

5. Place
   the gel mix in the autoclave and run on a setting that gets the sample to
   at least 121 °C under 20 psi for at least 30 min.

6. While
   your samples are sterilising in the autoclave, you shoµld
   prepare your plate pouring station:

- Find an empty
section of the lab bench with a working flame.

- Spray down the
bench with a 70% ethanol solution and wipe down with a paper towel.

- Count out the
appropriate number of plates and stack them on your lab bench.

- Label the
plates with the date and the medium they will contain including the identity of
the antibiotic.

- Prepare a
water bath at 60 °C with sufficient water to submerge ~75% of the bottle
containing your molten gel mixture.

7. Retrieve
   your molten agar mix from the autoclave.

8. Partially
   submerge your molten gel-mix in the 60 °C water bath for at least 5 mins.

9. Open one
   plate at a time next to the flame and begin pouring the control plate.

10. Light
    the flame at the plate pouring station and dilute 5 ml stock ampicillin
    into your ~60 °C molten gel mix using sterile technique.(
    final concentration: 100µg/ml)

11. Swirl
    the agar bottle to ensure even distribution of the antibiotic throµghout the agar.

12. Open one
    plate at a time next to the flame and begin pouring.

Note: Be sure to
swirl your plates after pouring to remove bubbles and ensure an even
distribution of agar over the bottom of the plate. Cap each plate after pouring
and stack as you pour.

13. Leave
    your plates out on the bench to solidify. at room temperature overnight to
    allow them to dry.

14. Place
    the plates in a plastic bag with an absorbent material to reduce
    condensation. The plates are then stored at 4 °C until use.

**2. Isolating Bacteria on an LB Agar Plate**

Chemicals required:

●
LB agar plate (with appropriate
antibiotic)

●
Bacterial stab/glycerol stock

Instruments required :

●
Pipette tip

●
Marker

Apparatus required:

●
Bunsen burner

●
Incubator

**Isolating Bacteria on an LB Agar Plate Protocol step:**

1. Obtain
   an LB agar plate with appropriate antibiotics.(our
   case: ampicillin)

2. Label
   the bottom of the plate with the plasmid name and the date.

3. Sterilise
   your lab bench by spraying it down with 70% ethanol and wiping it down
   with a paper towel. Maintain sterility by working near a flame or bunsen burner.

4. Obtain
   the appropriate bacterial stab or glycerol stock.

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

punctured area
of the stab cµlture or the top of the glycerol stock.

6. Gently
   spread the bacteria over a section of the plate, as shown in the diagram
   above, to create streak #1

7. Using a
   fresh, sterile toothpick, or freshly sterilised loop, drag throµgh streak #1 and spread the bacteria over a
   second section of the plate, to create streak #2.

8. Using a
   third sterile pipette tip, toothpick, or sterilised loop, drag throµgh streak #2 and spread the bacteria over the
   last section of the plate, to create streak #3.

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

![](https://static.igem.wiki/teams/4923/wiki/experimentdpic.png)

**3. Inoculating a Liquid Bacterial Cµlture**

Chemicals required:

●
Liquid LB

●
10 mg/mL ampicillin (from
stock)

●
LB agar plate with bacteria
colony

Instruments required :

●
Pipette

●
Pipette tip

●
Marker

●
Large centrifuge tube

Apparatus required:

●
Incubator

**Inoculating a Liquid Bacterial
Culture Protocol step:**

1. Prepare
   liquid LB.

2. Add
   10ml liquid LB to a tube, mix with 100µl Kanamycin.
3. (Or 2ml
   LB with 20µl Kanamycin)

4. Using a
   sterile pipette tip or toothpick, select a single colony from your LB agar
   plate.

5. Drop
   the tip or toothpick into the liquid LB + antibiotic and swirl.

6. Loosely
   cover the cµlture with sterile aluminium foil or
   a cap that is not airtight.

7. Incubate
   bacteria culture at 37°C for 12-18 hr in a shaking incubator.

**4. Plasmid Extraction**

Chemicals required:

●
Ethanol

●
Monarch® Plasmid Miniprep Kit

●
Monarch® Plasmid Miniprep
Columns

●
Monarch® DNA Elution Buffer

●
Monarch® Plasmid Wash Buffer 1

●
Monarch® Plasmid Wash Buffer 2

●
Monarch® Plasmid Resuspension
Buffer (B1)

●
Monarch® Plasmid Lysis Buffer
(B2)

●
Monarch® Plasmid Neutralization
Buffer (B3)

Instruments required :

●
Pipette

●
Pipette tip

●
Microfµge tube

Apparatus required:

●
Incubator

●
Centrifuge

**Before starting:**

Add 24 ml of ethanol to 6 ml of Monarch
Plasmid Wash Buffer 2.

Note that:

All incubations are under 35°C.

All centrifµgation
steps shoµld be carried out at 16,000 x g (~13,000
RPM) Store Plasmid.

Neutralization Buffer (B3) at 4°C after
opening.

**Plasmid Extraction Protocol:**

1. Pellet
   1.5 ml of cµlture bacterial cµlture
   (not to exceed 15 OD units) by centrifµgation
   for 30 seconds. Discard supernatants.

Ensure cµltures are not overgrown (12-16 hours is
ideal).

2. Resuspend
   pellet in 200 μl Plasmid Resuspension
   Buffer (B1) Vortex or pipet to ensure cells are completely resuspended.
   There shoµld be no visible clumps.

3. Lyse
   cells by adding 200 μl Plasmid Lysis Buffer
   (B2)

4. Invert
   the tube immediately and gently 5–6 times until colour changes to dark
   pink and the solution is clear and viscous. Do not vortex! Incubate for
   one minute.

5. Neutralise
   the lysate by adding 400 μl of Plasmid
   Neutralization Buffer (B3) Gently invert the tube until colour is
   uniformly yellow and a precipitate forms. Do not
   vortex! Incubate for 2 minutes.

6. Clarify
   the lysate by spinning for 3 minutes at 16,000 x g.

7. Carefµlly transfer supernatant
   to the spin column and centrifµge for 1 minute.
   Discard
8. flow-throµgh.

9. Re-insert
   column in the collection tube and add 200 μl
   of Plasmid Wash Buffer 1.

10. Incubate
    for 5 minutes, then Centrifµge for 1 minute.
    Discard the flow-throµgh.

11. Add 400
    μl of Plasmid Wash Buffer 2 and centrifµge for 1 minute.

12. Transfer
    column to a clean 1.5 ml microfµge tube. Use
    care to ensure that the tip of the column has not come into contact with
    the flow-throµgh.

13. Add 30 μl DNA Elution Buffer to the centre of the
    matrix. Wait for 1 minute, then spin for 1 minute to elute DNA.

**4. Bacterial Glycerol Stocks**

Chemicals required:

●
50% glycerol

●
Bacterial growth

Instruments required:

●
Pipette

●
Pipette tip

**Bacterial Glycerol Stocks Protocol:**

●
After you have bacterial
growth, add 500 μL of the overnight cµlture to 500 μL of 50%
glycerol in a 2 mL screw top tube or cryovial and gently mix.

●
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.

# Plasmid Resuspension From Its Powder Form

---

![](https://static.igem.wiki/teams/4923/wiki/experiment27.png)

---

**Experiment description:**

Resuspend
the plasmid to obtain a liquid solution containing the desired plasmid.

Chemicals required:

●
Plasmid

●
Distilled water

Instruments required :

●
Pipette

●
Pipette tip

Apparatus required:

●
Centrifuge

**Plasmid Resuspension Protocol:**

1. Centrifuge
   at 5,000×g for 5 min.

2. Carefully
   open the tube and add 20 μl of sterile
   water to dissolve the DNA.

3. Close
   the tube and incubate for 10 minutes at room temperature.

4. Briefly
   vortex the tube and then do a quick spin to concentrate the liquid at the  bottom.
   Speed is less than 5000×g.

5. Store
   the plasmid at -20 °C.

# Restriction Digestion and Ligation

---

![](https://static.igem.wiki/teams/4923/wiki/experiment26.png)

---

**Restriction Digestion**

**Experiment description:**

Using restriction enzymes to cut DNA into fragments in preparation
for further biological experiments (ligation).

Chemicals required:

●
EcoRI-HF

●
PstI-HF

●
10x rCutSmart
Buffer

●
DNA template

●
Distilled water

Instruments required :

●
Pipette

●
Pipette tip

●
Ice bucket

●
Microfµge tube

Apparatus required:

●
incubator

**Restriction Digestion Protocol:**

1. Thaw
   all reagents on ice.

2. Assemble
   reaction mix into 50 μL volume in a microfµge tube.

3. Add
   reagents in the following order: water, buffer, DNA template, restriction
   enzyme.

4. Gently
   mix by tapping the tube. Briefly centrifµge to
   settle tube contents.

5. (Prepare
   negative control reactions without template DNA. Prepare a positive
   control reaction with a template of known cutting sites corresponding to
   the restriction enzyme of choice).

6. Typical
   Incubation time and temperature is 37°C for 1 hour using the water bath.

OR

Incubate at 37°C for 5-15 minutes as
both enzymes are Time-Saver qualified.

**Ligation**

**Experiment description**:

Using
DNA ligase to join two DNA fragments throµgh the
formation of a phosphodiester bond.

Chemicals required:

●
Insert and vector DNA

●
T4 DNA ligase enzyme

●
Ligase buffer

●
Distilled/ Ionized water

Instruments required :

●
Pipette

●
Pipette tip

●
MicrofUge tube

Apparatus required:

●
Water Bath

**Ligation Protocol:**

1. Set up
   the following reaction in a microcentrifuge tube on ice.

2. Add
   reagents in following order: water, buffer, insert, vector, T4 ligase.

3. Gently
   mix the reaction by pipetting up and down and microfµge
   briefly.

4. For
   cohesive (sticky) ends and blunt ends, incubate at 16°C overnight using a
   water bath.

5. Heat the
   inactivation at 65°C for 10 minutes.

6. Chill on
   ice and transfer 1-5 μl using a
   micropipette of the reaction into 50 μl
   competent cells.

7. Insert:Plasmid

49.52 ng : 43.81ng (1:1) 99.04 ng : 43.81ng (2:1) 148.6 ng :
43.81ng (3:1)

# Bacterial Transformation

---

![](https://static.igem.wiki/teams/4923/wiki/experiment25.png)

---

**Experiment
description:**

Introducing a plasmid into a bacteria
to produce mµltiple copies of a recombinant DNA molecµle.

Chemicals required:

●
LB agar plate (with appropriate
antibiotic)

●
SOC media

●
Competent cells

●
DNA

Instruments required :

●
Pipette (5µl/50µl)

●
Pipette tip

●
Microcentrifµge tubes

●
Sterile spreading device

Apparatus required:

●
Incubator

●
Water bath

**Transformation Protocol:**

1. Take
   competent cells out of -80°C and thaw on ice (approximately 20-30 mins).

2. Remove
   agar plates (containing the appropriate antibiotic) from storage at 4°C
   and let them warm up to room temperature and then incubate in a 37°C
   incubator.

3. Mix 5 μl of DNA (usually 10 pg
   - 100 ng) into 50 μl of competent cells in
   a microcentrifµge GENTLY mix by flicking.

4. Incubate
   the competent cell/DNA mixture on ice for 20-30 mins.

5. Heat
   shock each transformation tube by placing the bottom 1/2 to 2/3 of the
   tube

into a 42°C
water bath for 1 min.

6. Put the
   tubes back on ice for 2 min.

7. Add 950
   μl SOC media (without antibiotic) to the
   bacteria and grow in a 37°C incubator for 1 hour.

8. Plate
   100µl of the transformation onto a 10 cm LB agar plate containing the
   appropriate antibiotic.

9. Incubate
   plates at 37°C overnight.

# Protein Induction

---

![](https://static.igem.wiki/teams/4923/wiki/experiment23.png)

---

**Experiment description:**

Add
protein inducer to stimµlate
the expression of the target gene according to the T7 promoter.

Chemicals required:

●     LB

●
IPTG

●
Stock kanamycin (10mg/ml)

Instruments required:

●
Pipette (5µl/50µl)

●
Pipette tip

●
Microcentrifuge tubes

●
Erlenmeyer flask

Apparatus required:

●
Incubator

●
Shaker

●
Centrifuge

**Protein Induction Protocol:**

1. Make a
   fresh plate for better protein expression. Pick a colony, Resuspend a positive
   colony from the transformation in 5 ml LB broth medium with 25µl kanamycin.(from stock) Incubated and shake at 30 °C and
   250 rpm overnight

2. Next
   morning, inocµlate LB+ Antibiotic (25ml) in
   250ml Erlenmeyer flask with 250µl of bacteria containing solution (1:100)
   or inocµlate enoµgh
   expression cµlture for the initial OD to be 0.1.

3. Shake
   at 30 °C and 250 rpm /3-4 hours at 37°C in a rotator. Until OD600 reached
   0.4- 0.8

(OPTIONAL: When
OD reaches 0.5-0.6, reduce incubator temperature to 16°C).

4. Prepare
   60 ml LB + Antibiotic + 1mM IPTG and prewarm to 37°C about 10 minutes
   before use. (Thaw IPTG on ice for at least the first few minutes.)

(Dissolve
0.0143g of IPTG with 59.7ml LB and 300µl kanamycin from stock)

5. After
   growing for 3-4 hours, remove 1 ml from tubes prepared in step 3 and place
   in labelled 1.5 ml tubes. Centrifµge at maximum
   speed for 30 seconds at room temperature and remove supernatant. Freeze
   pellets at -20°C until needed. THIS IS THE UNINDUCED CONTROL.

6. Add
   24.25 ml ml prewarmed (37°C) LB + Antibiotic +
   1mM IPTG prepared in step 4 to tubes prepared in step 3 (to a final
   concentration of 0.5 mM)

7. Shake
   for 5 hours at 30°C or overnight (16-18h) at 16°C at
   250rpm

8. Harvest
   cells by centrifµgation at 20,000g and 4°C for
   20 minutes

(Harvest in
round-bottomed 50 ml centrifµge tubes and use a centrifµge machine)

Or

9. Transfer
   1 ml from the induced sample to labelled 1.5 ml tubes and centrifµge at maximum speed for 30 seconds at room
   temperature and remove the supernatant.

10. Freeze
    pellet at -20°C until needed

# Bacterial Lysis

---

![](https://static.igem.wiki/teams/4923/wiki/experiment22.png)

---

**Experiment description:** Breaking the cell membrane to obtain the desired protein from the bacteria.

ChemicalS required:

* B-PER bacterial Protein Extraction Reagent (in phosphate buffer)
* 40mL bacterial cµlture, A600 = 1.5-3.0

**Protein Extraction Protocol:**

1. 1.Centrifugation at 5000 × g for 10 minutes and discard the supernatant.

1. Add 5mL of B-PER Reagent to the pellet and pipetting up and down until it is clear.

(Note: bacteria solution 8 : 1 lysis buffer)

1. Gently shake the suspension for 10 minutes.

1. Centrifµgate at 15,000 × g for 15 minutes. Transfer supernatant to a new tube which contain the protein required

# Protein Purification

---

![](https://static.igem.wiki/teams/4923/wiki/experiment21.png)

---

**Experiment description:**

Using protein a to bind with to obtain
highly pure, stable and active protein

Chemicals required:

●
Protein
A IgG Purification Kit:  Protein A column, elution buffer, binding
buffer.

●
Antibody
Sample: Centrifµge cloudy samples and use only the
clear supernatant.

**Purification of IgG** **Protocol:**

1. Sequentially
   remove top first and bottom cap second from Protein A column.

2. Pour
   off and save the storage solution in a tube and cap the tube.

3. Apply
   3-5mL of Binding Buffer and allow it to drain throµgh
   the column.

(Note: Do not leave the drained
column uncapped for more than a few minutes.)

4. Apply
   up to 5mL of Antibody Sample to the column and allow it to flow throµgh the column

5. Add
   5-15mL of Binding Buffer and allowing it to drain throµgh
   the column

6. Add
   5-10mL of the IgG Elution Buffer to the column, collect separate 1mL
   fractions of the solution that drains from the column.

7. Determine
   which fractions contain antibodies by measuring the absorbance of each one
   at 280 nm and comparing it to the neutralised Elution Buffer alone.

            ( Note:
fractions 2, 3 and 4 usually have the highest absorbance values.)

**Storage
of Protein A Column**

1. Wash
   the column with 5-10mL of Elution Buffer. Columns may be regenerated a
   minimum of 10 times without significant loss of binding capacity.

2. Add a storage solution to the column and
   replace the bottom cap on the column followed by the top cap. Store the
   column upright at 4°C.

# Elisa Assay

---

![](https://static.igem.wiki/teams/4923/wiki/experiment27.png)

---

**Label
primary antibody with HRP**

**Experiment description:**

Conjugation
of antibody with HRP for the ELISA Kit

Chemicals required:

●      HRP Conjugation Kit - Lightning-Link®

●      Antibody

●      Modifier reagent

●      Quencher reagent

Instruments required:

●
10ul/100ul pipettes

●
Pipette tips

Apparatus required:

●
Incubator

●
Vortex

**Labelling Antibody Protocol:**

1. Add 10 μL
of Modifier reagent to 100 μL of antibody to be
labelled and mix gently.

2. Pipette the antibody sample directly onto the vial of HRP Conjugation
Mix. Pipette up and down 1-3 times afterwards

3. Replace cap on the vial and leave it standing for 3 hours or more in
the dark at room temperature. (Note: Longer incubation times, such as overnight, have no
negative effect on the conjugation.)

4. After incubating for 3 hours (or more), add 10 μL
of Quencher reagent and mix gently. The conjugate can be used after 30 minutes.

**Direct ELISA Kit**

**Experiment description:**

Testing
whether the antibody can bind with the antigen by determining the wavelength

Chemicals required:

●      Coating Buffer

●      Tris Buffered Saline (TBS)

●      Wash Solution

●      Blocking (Postcoat)
Solution

●      Conjugate Diluent

●      0.18 M H2SO4

Instruments required:

●      100ul/ 200ul pipettes

●      Pipettes tip

●      ELISA plate

Apparatus required:

●
Incubator

●
Plate reader

Experimental flow:

1. Coat with Antigen

2. Blocking (Postcoat)

3. HRP Conjugated Primary
Antibody

4. Enzyme Substrate Reaction

**1. Coat with Antigen**

1. Transfer 100 ul of 1 – 2 ug/ml purified antigen to each well.

2. Incubate coated plate for 60 minutes.

3. After incubation, remove the antigen solution from each well.

4. Wash each well with Wash Solution as follows:

● Fill each well with Wash Solution

● Remove Wash Solution by pipette

 ● Repeat for a total of 3
washes.

**2. Blocking (Postcoat)**

1. Add 200 ul of Blocking (Postcoat) Solution
to each well.

2. Incubate 30 minutes.

3. Remove the Blocking (Postcoat) Solution and
wash each well 3 times.

**3. HRP Conjugated Primary Antibody**

1. Add the HRP Conjugated Primary Antibody in Conjugate Diluent

2. Transfer 100 ul to each well.

3. Incubate 60 minutes.

4. Remove HRP Conjugate and wash each well 5 times.

**4. Enzyme Substrate Reaction**

1. Transfer 100 ul of substrate solution to each well.

2. Incubate plate 5 - 30 minutes.

3. Apply 100 ul of 0.18 M H2SO4 to each well.

4. Using a microtiter plate reader, read the plate at 450 nm wavelength
for the substrate.

RETURN TO THE TOP

## Experimental Protocols

Polymerase Chain Reaction
Agarose Gel Electrophoresis
Plasmid Extraction
Plasmid Resuspension From Its Powder Form
Restriction Digestion and Ligation
Bacterial Transformation
Protein Induction
Bacterial Lysis
Protein Purification
Elisa Assay

{% endblock %}

{% extends "layout.html" %}
{% block title %}Results{% endblock %}
{% block lead %}{% endblock %}
{% block page\_content %}

## (I) PCR

---

![](https://static.igem.wiki/teams/4923/wiki/results3839.jpg)

After PCR, we carried out Gel Electrophoresis. Our J591 gene sequence is ~ 2800 bp. In
Figure1, according to the ladder, it suggested there is an obvious banding for the gene of
J591 lines between the base pair from 2kb to 3kb. Our genes were successfully amplified.

## (II) Ligation

---

![](https://static.igem.wiki/teams/4923/wiki/results3840.jpg)

After ligation, we carried out single digestion to produce recombinant plasmid with around
8000-9000 bp. In Figure 2, according to the ladder in the left, it is suggested that there is an
obvious banding of the gene of recombinant plasmid between the base pairs between 8kb to
9kb. We successfully insert the target gene to the plasmid.

## (III) Antibody concentration

---

![](https://static.igem.wiki/teams/4923/wiki/results-iii.png)

We checked the concentration of the antibody in different samples by measuring the absorbance A260 / A280 using a spectrophotometer. The graph shows that mean antibody concentration under different concentrations of IBTG. 0.4 mM IBTG induced highest protein expression.

## (IV) Elisa kit

---

Elisa kit is a biochemical assay tool used to detect and quantify specific protein, antibody and peptides. We labeled our sample with a type of enzyme called HRP by targeting primary amine groups which is present in IgG antibodies. For other proteins that cannot bind with HRP, the enzyme will be deactivated by the quencher and removed during the first washing. After that we add the conjugated antibody solution to the ELISA plates with antigens coating on it, then substrate. To prevent the binding of any non-specific antibodies to the plate and minimize false-positive results, we washed the plate several times to remove the non-binding protein. The color change occurred due to the reaction between HRP bind to the antibody and substrate and we read the absorbance of the mixture of 450 nm wavelength.

However, the PSMA antigen of 50ug cost HK$2200, as an IGEM team with cost efficiency in mind, we needed to carefully allocate our resources to minimize our expenditure. As the amount of antigen added to each vial of ELISA plate is fixed, ranging from 1-2ug/ml, we chose the minimum concentration of the antigen to limit the consumption of the antigen. Moreover, by using low concentration of PSMA in ELISA kit, it can simulate the condition of prostate cancer in the early stages. We can thus obtain data that are similar to ones collected from an actual patient.

According to the protocol of the ELISA KIT, it is recommended that the antibody level can be
determined using 450 nm wavelength checked by the microplate reader. The results were
interpreted using SPSS statistical analysis by independent sample T-test. Water was used
instead of the J591 antibodies we synthesized as the control. There is a significant
difference in the mean value of 450 nm between water and J591 antibody. The mean
absorbance for J591 antibody and water is 2.8462 and 0.1102 respectively (P < 0.01). It is
found that the mean absorbance was 20 times folder larger in J591.

![](https://static.igem.wiki/teams/4923/wiki/results3841.jpg)

There is an obvious color difference in the plate for water sample and J591 sample under
the ELISA KIT.

![](https://static.igem.wiki/teams/4923/wiki/results3842.jpg)

## (V) Optimization of Antibody Concentration

---

We use non-linear regression to work out the minimal amount of the concentration to
determine the fixed amount of the antigen. In order to determine the 1 ug / ml, the optimal
concentration for the antibody is around 2.0 ug / ml from the result shown below. It passes
the non-linear regression with p-value (p < 0.05).

![](https://static.igem.wiki/teams/4923/wiki/results.png)

![](https://static.igem.wiki/teams/4923/wiki/results3844.jpg)

{% endblock %}