Experiments | BZK - iGEM 2023

Experiments

The plasmid construction of pET28a-UAO 、 pET28a- allantoinase 、 pET28a- allantoicase

Preparing Culture medium

Materials:

Tryptone , Yeast extract, ddH ₂ O, NaCl, Digital balance

Procedure:

A: Ensure the volume of culture medium required is 500ml, and the concentrations required for NaCl, tryptone and yeast extract are 10g/L, 10g/L and 5g/L.

Reagent	Volume/mass
Tryptone	5g
Yeast extract	2.5g
Ddwater	500ml
NaCl	5g

B: Measure 100ml ddwater with measuring cylinder. Add the water perceived into a flask.

C: Use digital balance to measure 5g of NaCl sample. Set the sample on a weighing paper and add solid sample to the balance via spatula. After measuring, add the sample in the flask.

D: repeat the procedure with measurement of 5g tryptone sample.

E: repeat the procedure with measurement of 2.5g yeast extract sample.

F: Seal the flask with culture medium with tape. Label it as “LB Culture” and prepare for its further sterilization.

Amplify the Target Gene PCR （ UAO 、 Allantoinase and Allantoicase ）

2.1 PCR

Materials

UAO, PCR thermal cycler, Allantoinase, Allantoicase, primer, Mix

Procedure I

A: Measure 10μl of mix by pipette and add it to the PCR tube.

B: Measure 7μl of ddwater by pipette and add it to the PCR tube.

C: Measure 1μl of primer R1 and 1μl of primer F1 by pipette to the PCR tube.

D: Measure 1μl of template by pipette and add it to the PCR tube.

E: Fully mix the substances in the tube by attaching the PCR tube to the vortex.

*The sequence of adding substances is arranged by their gradient in amounts.

UAO(981bp)	Amount	Allantoinase(1485bp)	Amount	Allantoicase(1228bp)	Amount
2mix	10μl	2mix	10μl	2mix	10μl
UAO-R1	1μl	A-nase-R1	1μl	A-case-R1	1μl
UAO-F1	1μl	A-nase-F1	1μl	A-case-F1	1μl
UAO template	1μl	Allantoinase template	1μl	Allantoicase template	1μl
ddwater	7μl	ddwater	7μl	ddwater	7μl
total	20μl	total	20μl	total	20μl

*2 mix: Since the product possesses volume of 20 μl, the amounts of substances in “mix”. should be doubled so that the concentration is constant.

*mix contains DNA polymerase, dNTP(dATP, dCTP, dGTP, or dTTP), buffer, Mg2+ ions.

*bp refers to the number of base groups contained in a DNA chain

Procedure II:

PCR program	Temp. (celsius degrees)	Period
Pre-denaturation	95	3min.
Denaturation	95	30sec.
Annealing	55	30sec.
Elongation	72	2min.
Elongation	72	30sec.

2.2 Agarose Gel Electrophoresis

Materials

Agarose, TAE1, Nucleic acid dye

Procedure

A: preparation of gel:

Ensure that the volume required is 100ml.

Prepare a flask. Use measuring cylinder to measure 100ml TAE and add it to the flask. Use digital balance and weighing paper to measure 1.5g agarose sample and add it to the flask.

Heat the flask at high temperature for 3 minutes in microwave oven.

Add 2μl nucleic acid dye into the flask.

*The theoretic amount of agarose required is 1g; however, the result that the gel fails to form solid structure and there’re regions remained uncoagulated demonstrates that more agaroses are needed. Consequently, we increase its mass by 50%.

B: Add the gel into the gel box. Wait for approximately 10 minutes, when it fully congeals and forms jelly-like substance.

C: Use pipette to measure 7μl product from the PCR program(it originally possesses 20μl), and add it to the well comb of the apparatus for electrophoresis.

D: Add 10μl DNA marker for reference of the categorization for the molecules separated.

E: Electrophoresis: connect the apparatus to the power supply with 180V for 15 minutes.

F: sever the connection to the power supply. Place the gel in the Blue LED transilluminator ， and observe it under the illumination of ultraviolet radiation.

RESULT: the signs that refer to PCR samples for each group approximately correspond to the marker(according to the length of templates).

2.3 Recovery of the products of PCR

*purpose: the product of PCR contains several different substances, while the replicated DNA is the required section. They are thus extracted from the product.

Materials

Buffer B2, Product of PCR

Procedure

A. Obtain a centrifuge tube which has a volume of 1.5ml. Use pipette to measure 300μl buffet b2 and add it to the tube.

B. Use pipette to measure 13μl from the product of PCR and add it to the centrifuge tube.

*13μl is the volume of product of PCR remaining after electrophoresis.

C. Transfer the mixture in the centrifuge tube to the absorption column. Attach it to a centrifuge shield and place it in the centrifuge(machine), start the centrifuging process with centrifugal force of 8000 xg and time of 30 seconds. Remove the mixture in the centrifuge shield after the process.

D. Use pipette to add 500μl wash buffer into the absorption column and start the centrifuging process with centrifugal force of 9000 xg and time of 30 seconds. Remove the mixture in the centrifugal shield.

E. Repeat procedure D

F. Without adding any substance, start centrifuging with centrifugal force of 9000 xg and time of 1 minute.

G. Obtain a new centrifuge tube, add 30μl Elusion Buffer to it via pipette, place it at rest for 1 minute, then centrifuge it with 9000 xg for 1 minute. After that, preserve the centrifuge tube.

H. Measure the concentration of the remaining mixture: by using spectrophotometer

-Use ddwater to rinse the detector of the apparatus.

-add two samples of ddwater as control group: for the situation of adding nothing(pure solvent). The result is close to zero.

-Add sample to the detector.

-the concentration of five known samples from the experiment: 22.65; 24.35; 27.05; 16.35; 20.2. (unit: ng/μl)

Plasmid extraction and enzyme digestion plasmid

3.1 Extraction of Plasmids

Materials

Buffer SP1,Buffer SP2,Buffer SP3

Procedure

A. Use pipette to measure 2ml of bacteria solution and transfer it to a 2ml centrifuge tube(use pipette with maximum range of 1000μl and operate twice). Place the centrifuge tube into the centrifuge(machine), and then start centrifuging with centrifugal force of 12000 xg and time of 1 minute.

*purpose: To separate the cells and medium in the bacteria solution.

B. Use pipette to measure 250μl of buffer SP1, add it to the centrifuge tube in procedure A. Continuously inject and retrieve the liquid in the pipette, which separates the bacteria aggregated at the bottom of the tube and spread them till they suspend/levitate in the solution.

*purpose: To spread the bacteria in the tube.

C. use pipette to measure 250μl of buffet SP2 and add it to the centrifuge tube.

*purpose: to crack the cell membranes of the bacteria, which exposes the plasmids to the solution.

D. Use pipette to measure 350μl of buffet SP3 and add it to the centrifuge tube.

*purpose: to neutralize buffet SP2.

E. Centrifuge the product in procedure D with centrifugal force of 12000 xg and time of 5 minutes. After that process, it can be observed that white, solid substances remain at the bottom of the tube.

*purpose: to separate the organelles and remnants of cell membranes with the plasmids.

F. Use pipette to measure 700μl of supernatant in the centrifuge tube and transfer it to the absorption column, then centrifuge it with force of 9000 xg and time of 1 minute.

G. Use pipette to measure 500μl wash buffer and add it to the absorption column. Centrifuge it with force of 9000 xg and time of 30 seconds. Repeat this procedure again.

H. Centrifuge the product of procedure G without adding any agent. The indices are 9000 xg and 1 minute.

I. Obtain a new centrifuge tube. Use pipette to measure 30μl Elution Buffer and add it to the absorption column. Place the tube at rest for a minute, and then centrifuge it with indices of 9000 xg, 1 minute. The remnant in the centrifuge tube is the vector required.

3.2 Enzyme Cutting

Materials

pET28a, template, Enzyme

Procedure

A.Ensure that to aggregate volume required is 20μl or 50μl. The reagents are added according to the gradient of their volumes.

*the following values are corresponding to the group which cuts allantoinase.

B.Use pipette to measure 10μl of pET28a sample created from vector extraction. Transfer it to the centrifuge tube.

C.Use pipette to measure 6μl of ddwater and transfer it to the centrifuge tube.

D.Use pipette to measure 2μl of Cut smart Buffer and transfer it to the the centrifuge tube.

E.Use pipette to measure 1μl of BamHI restriction enzyme and transfer it to the centrifuge tube.

F.Use pipette to measure 1μl of NotI restriction enzyme and transfer it to the centrifuge tube.

G.Attach the tube to the vortex.

H.Set the tube to a 37 Celsius Degree environment for 30 minutes.

*It’s the optimum temperature for the restriction enzymes.

*It’s the procedure for enzyme cutting.

I. Store the sample in ice.

J. Repeat procedure A to I while replacing the pET28a vector to allantoinase (target. gene. )

Group of UAO:

Vector system	Amount/μl	Target gene system	Amount/μl
PET28a	10	UAO	10
Cut smart buffer	2	Cut smart buffer	2
BamHI	1	BamHI	1
Xhol	1	Xhol	1
Ddwater	6	Ddwater	6
total	20	total	20

Group of a-nase:

Vector system	Amount/μl	Target gene system	Amount/μl
PET28a	10	allantoinase	10
Cut smart buffer	2	Cut smart buffer	2
BamHI	1	BamHI	1
NotI	1	NotI	1
Ddwater	6	Ddwater	6
total	20	total	20

Group of a-case:

Vector system	Amount/μl	Target gene system	Amount/μl
PET28a	30	allantoicase	30
Cut smart buffer	5	Cut smart buffer	5
SacI	1	SacI	1
Xhol	1	Xhol	1
Ddwater	13	Ddwater	13
total	50	total	50

3.3 Electrophoresis

* The product of enzyme cutting for target genes cannot be examined, since they can’t be separated by certain features.

Materials

Loading buffer, Marker,Vector samples-control group

Procedure

A.Use pipette to measure loading buffer and add it to the centrifuge tube which contains severed pET28a. The volume required for the buffer is 10% of the total volume of the vector sample. (e.g. for allantoinase group, since the volume of the vector is 20μl, the loading buffer added is 2μl)

B.Add samples to the well comb: two samples of 10μl marker, 20μl of control group sample, and 20μl for each kind of sample.

C.Connect to 160V electricity for approximately 20 minutes

Enzyme ligation and transformation

4.1 LB Agar Plates

Materials

Pre-prepared liquid sterilized agar solution, Agar powder

Procedure

a) Acquired sterilized liquid agar solution

b) Put all materials inside of fume hood workstation; everyone working under fume hood sterilized hands with ethanol

c) Light an alcohol lamp within fume hood

d) Add 3g of agar powder to liquid agar solution and mix

e) Prepare a 50mL centrifugal tube, pour some of the prepared mixture and add KANT sample

f) Pour previously mentioned mixture with KANT evenly onto petri dish, make sure it spreads throughout the dish and remove any air bubbles

g) Set aside petri dish with lid awry to cool and solidify

4.2 Ligation Process

Materials

pET28a vector,UAO/a-nase/a-case 12μl,T4 ligase

Procedure

A.Ensure that the volume required for the product of ligation system is 20μl

B.Obtain a centrifuge tube(1.5ml), use pipette to measure 12μl product of target gene(undergone enzyme cutting) and transfer it to the tube.

C.use pipette to measure 3μl product of pET28a(undergone enzyme cutting) and transfer it to the tube.

*The proportion between volume of gene and vector is 4:1 or 6:1.

D.use pipette to measure 2μl of 10xT4 ligase and transfer it to the tube.

E.use pipette to measure 2μl ddwater and transfer it to the tube.

F.use pipette to measure 1μl of T4 ligase and transfer it to the tube.

G.Attach the tube to the vortex.

H.Set the tube at rest under room temperature for 30 minutes.

Reagent	Volume/mass
pET28a vector	3μl
UAO/a-nase/a-case	12μl
T4 ligase	1μl
10xT4 buffer	2μl
Ddwater	2μl

4.3 Transformation of Bacteria

Materials

Ligated samples, LB Agar plates and liquid solution

Procedure

a)Ligated samples are added to E.Coli samples

b)Placed on ice for 20 minutes.

c)Heat at 42 degrees Celsius for 45 seconds, then immediately put on ice for 2-3 minutes.

d)Add 900μL LB Agar solution, incubate for 30 minutes

e)Centrifuge at 5000xg for 3 minutes, extract 900μL of supernatant then discard

f)Apply end product to agar plates, incubate for 12-16hr at 37 degrees Celsius.

*Possible mistake: It’s required to spread bacteria mildly, since harsh physical shock will damage their structure.

4.4 Isolated colonies validation

*purpose: DH5alpha bacteria containing constructed plasmids has multiplied. Isolated colonies validation is used for ensuring that the plasmids have replicated during this process.

Procedure

a)Within sterilized fume hood workstation, remove a colony of preprepared bacteria from the agar plate and add to 1.5mL centrifuge tube (Note: choose larger colonies but also be aware that colonies near the sides of the petri dish are most likely to be contaminated or are undesired bacteria and are less ideal)

b)Using pipette, add the 10µL mixture to the PCR tube.

c)Using pipette, add the 1µL F primer to the PCR tube.

d)Using pipette, add the 1µL R primer to the PCR tube.

e)Using pipette, add the 8uL of ddH2O to the PCR tube.

f)Attach the tube to the vortex, then place it to the PCR machine.

PCR program	Temp. (celsius degrees)	Period
Pre-denaturation	95	3min.
Denaturation	95	30sec.
Annealing	55	30sec.
Elongation	72	2min.
Repetition-Denaturation,Annealing,Elongation	/	30times

Procedure

A. Obtain a conical flask. Use measuring cylinder to measure 100ml of (1x)TAE solution and transfer it to the conical flask.

B. Place the agarose sample on the weighing paper, Use digital balance and spatula to measure 1g of agarose sample and add it to the flask.

C. Place the flask in the microwave oven and heat at high temperature for 2 to 3 minutes.

D. Use pipette to measure 5μl of Nucleic acid dye(Gel red) and add it to the flask.

E. Add the sample in the flask to the mode of electrophoresis instrument.

F. Wait for approximately 20 minutes till the gel coagulates.

Homologous Recombination （ pGex-4T-1-UAO 、 pGex-4T-1-UAA ）

1. PCR pGex-4T-1, Uric acid oxidase 、 allantoinase and allantoi C ase

Procedure

For vectors pGex-4T-1:

REAGENT	AMOUNT/μl
Primer start	10
pGex-4T-1 primer F	1
pGex-4T-1 primer R	1
pGex-4T-1 template	1
DdH2O	7
total	20

For target gene UAO ：

REAGENT	AMOUNT/μl
2mix	10
UAO primer F	1
UAO primer R	1
UAO template	1
DdH2O	7
total	20

For target gene Allantoinase ：

REAGENT	AMOUNT/μl
2mix	10
A-nase primer F	1
A-nase primer R	1
A-nase template	1
DdH2O	7
total	20

For target gene Allantoicase ：

REAGENT	AMOUNT/μl
2mix	10
A-case primer F	1
A-case primer R	1
A-case template	1
DdH2O	7
total	20

Procedures

For vectors pGex-4T-1:

PCR program	Temp. (celsius degrees)	Period
Pre-denaturation	95	3min.
Denaturation	95	30sec.
Annealing	55	30sec.
Elongation	72	3min.
Repetition-Denaturation,Annealing,Elongation	/	30times
Elongation	72	10min.

For gene sections(UAO/A-nase/A-case):

PCR program	Temp. (celsius degrees)	Period
Pre-denaturation	95	3min.
Denaturation	95	30sec.
Annealing	55	30sec.
Elongation	72	2min.
Repetition-Denaturation,Annealing,Elongation	/	30times
Elongation	72	10min.

2. Electrophoresis

Procedure

A. According to the volume of the vectors and genes samples, use pipette to measure certain amount of loading buffer to the PCR tubes containing the samples.

*Volume of sample in PCR tube : Volume of the loading buffer is 10 : 1

Consequently, since volume for the remaining sample of vector is 20μl and the volume for the remaining sample of target genes is 5μl, the volume of loading buffer added to each sample are pertinently 2μl and 0.5μl.

B. Use pipette to measure 10μl of marker and add it to the well comb in the electrophoresis instrument.

C. Use pipette to measure 7μl for each sample of vector pGex-4T-1 and target gene UAO, Allantoinase , and Allantoicase in the well combs of the electrophoresis instrument.

D. Connect the instrument to a 180V power source for approximately 25 minutes.

E. Place the sample under the illumination of the ultraviolet radiation, then observe the results.

3. Extraction

Procedure

A.Use pipette to measure 2 samples of 300μl Buffer B2 and transfer them separately to two labelled centrifuge tube.

B.Use pipette to measure 15μl of vector sample and 15μl of target gene sample.

C.Transfer the mixture in the centrifuge tubes to the absorption columns. Attach them to centrifuge shields and place them in the centrifuge(machine), start the centrifuging process with centrifugal force of 8000 xg and time of 30 seconds. Remove the mixtures in the centrifuge shields after the process.

D.Use pipette to add 500μl wash buffer into the absorption columns and start the centrifuging process with centrifugal force of 9000 xg and time of 30 seconds. Remove the mixtures in the centrifugal shields.

E.Repeat procedure D.

F.Without adding any substance, start centrifuging with centrifugal force of 9000 xg and time of 1 minute.

G.Obtain 2 new centrifuge tubes, add 30μl Elusion Buffer to it via pipette, place them at rest for 1 minute, then centrifuge them with 9000 xg for 1 minute. After that, preserve the centrifuge tubes.

H.Measure the concentration of the samples received.

4. pGex-UAO and pGex-UAA homologous recombination

Materials

DpnI enzyme sample,PCR product: vector pGex-4T-1,PCR product: target genes(UAO, A-nase, and A-case)

Procedure I

A.Ensure the volume of each PCR sample and determine the volume of DpnI enzyme sample required for the system. The volume of each sample in the experiment is 30μl, indicating the volume required for enzyme is 1μl for each.

*The DpnI enzyme prevent the DNA molecules from methylation.

*The theoretical relationship of value between the sample and DpnI needed: 10μl of sample requires 1μl DpnI, 30μl of sample requires 2μl DpnI. However, since the concentration of PCR samples is relatively low, less DpnI is needed.

B.After adding the ppnI enzyme sample, place the PCR tube in 37 Celsius Degree environment for 10 minutes.

C.Place the PCR tubes in 80 Celsius Degree environment for 20 minutes.

*Excess DpnI will deform the structure of DNA molecules. High temperature of 80 Celsius Degrees will denature them fully.

Procedure II

Every group constructs a pGex-UAO plasmid and a pGex-UAA plasmid. Since the concentration of vector samples used is inconsistent, the volume of reagents used in each group varies. (Follow certain formula)

For pGex-UAO:

Reagents	amount
(total)	10μl
DNA recombinase	5μl
Vector: pGex-4T-1	1.5μl
UAO-2	1.0μl
ddwater	2.5μl

For pGex-UAA:

Reagents	amount
(total)	20μl
DNA recombinase	5μl
Vector: pGex-4T-1	1μl
UAO-2	1μl
A-nase	2μl
A-case	10μl
Ddwater	1μl

Procedure III

1.The UAO group: Place it in 50 Celsius Degree surrounding for 5 minutes. Then place it in 4 Celsius degree environment.

2.The UAO group: Place it in 50 Celsius Degree surrounding for 15 minutes. Then place it in 4 Celsius degree environment.

5. Transformation of Bacteria

Procedure

a)Ligated samples are added to E.Coli(DH5) samples

b)Placed on ice for 20 minutes.

c)Heat at 42 degrees Celsius for 45 seconds, then immediately put on ice for 2-3 minutes.

d)Add 900μL LB Agar solution, and incubate for 30 minutes

e)Centrifuge at 5000xg for 3 minutes, extract 900μL of supernatant then discard

f)Apply end product to agar plates, incubate for 12-16hr at 37 degrees Celsius.

*Possible mistake: It’s required to spread bacteria mildly, since harsh physical shock will damage their structure.

6. Isolated colonies validation

*purpose: examine the vectors(pGex-UAO and pGex-UAA) in the bacteria DH5alpha

Procedure I

A. Use pipette to measure 8 of ddwater and add it to the PCR tube.

B. Obtain template: using a 0.1~2.5μl pipette to mildly scratch the plate with bacteria and add it to the PCR tube. (For two vectors, pGex-UAO and pGex-UAA, operate separately )

C. Use pipette to measure 10μl of 2mix and add it to the PCR tube.

D. Use pipette to measure 1μl of UAO-2-F or UAA-F and add it to the PCR tube.

E. Use pipette to measure 1μl of UAO-2-R or UAA-R and add it to the PCR tube.

F. Attach the PCR tubes to the vortex.

Reagents for pGex-UAO

Reagents	Amount/μl
2mix	10
UAO-2-F	1
UAO-2-R	1
pGex-UAO	(0)
ddwater	8

Reagents for pGex-UAA

Reagents	Amount/μl
2mix	10
UAA-F	1
UAA-R	1
pGex-UAA	(0)
ddwater	8

Procedure II-PCR process

PCR program	Temp. (celsius degrees)	Period
Pre-denaturation	95	3min.
Denaturation	95	30sec.
Annealing	55	30sec.
Elongation	72	3min.
Repetition-Denaturation,Annealing,Elongation	/	30times
Elongation	72	10min.

Procedure III-electrophoresis

A.Add samples to the well comb: two samples of 10μl marker and 13μl of the samples in each group.

B.Connect to 160V electricity for approximately 20 minutes.

C.Examine the result with the illumination of ultraviolet radiation.

After the examination, shake the bacteria of DH5alpha containing target vectors in 37 Celsius Degree for 12~16 hours with adding LB to the tubes.

*Purpose: increase the amount of target vectors and prepare for transformation.

Incubate the BL21(DE3)

7.1 Plasmid extraction

Procedure

A. Use pipette to measure 2ml of bacteria solution and transfer it to a 2ml centrifuge tube. Place the centrifuge tube into the centrifuge(machine), and then start centrifuging with centrifugal force of 12000 xg and time of 1 minute.

C. use pipette to measure 250μl of buffet SP2 and add it to the centrifuge tube.

D. Use pipette to measure 350μl of buffet SP3 and add it to the centrifuge tube.

F. Use pipette to measure 700μl of supernatant in the centrifuge tube and transfer it to the absorption column, then centrifuge it with force of 9000 xg and time of 1 minute.

G. Use pipette to measure 500μl wash buffer and add it to the absorption column. Centrifuge it with force of 9000 xg and time of 30 seconds. Repeat this procedure again.

H. Centrifuge the product of procedure G without adding any agent. The indices are 9000 xg and 1 minute.

7.2 Transformation

*purpose: a necessary prerequisite for the following steps: the examination of vectors in BL21 and the protein synthesis.

*Method: Heat shock

Materials

pGex-UAO, pGex-UAA,BL21(DE3)

Procedure

a)Add vector samples to the bacteria sample.

b)Placed on ice for 20 minutes.

c)Heat at 42 degrees Celsius for 45 seconds, then immediately put on ice for 2-3 minutes.

d)Add 900μL LB Agar solution, incubate for 30 minutes

e)Centrifuge at 5000xg for 3 minutes, extract 900μL of supernatant then discard

f)Apply end product to agar plates, incubate for 12-16hr at 37 degrees Celsius.

8. Isolated colonies validation and shaking bacteria

*purpose: to replicate the vector and BL21, prepare for protein synthesis

Procedure I

A. Pick a 1.5ml centrifuge tube, use pipette to add 8μl ddwater to the tube.

B. Use 0.1~2.5μl pipette to obtain BL21 from the plate and add it to the tube.

C. Attach the tube to the vortex to form the bacteria solution

D. Use pipette to measure 2μl of the bacteria solution and add it to a new, 50ml centrifuge tube.

E. Use pipette to measure 900μl of LB culture medium and add it to the centrifuge tube.

F. Place the centrifuge on the shaking bed.

(The procedure is the same as the STEP II)

Procedure II

1.Procedure-Construct PCR system

A. Use pipette to measure 8μL of ddwater and add it to the PCR tube.

B. Obtain template: Add the sample of BL21 into the PCR tube

C. Use pipette to measure 10μl of 2mix and add it to the PCR tube.

D. Use pipette to measure 1μl of UAO-2-F or UAA-F and add it to the PCR tube.

E. Use pipette to measure 1μl of UAO-2-R or UAA-R and add it to the PCR tube.

F. Attach the PCR tubes to the vortex.

Procedure III-PCR process

PCR program	Temp. (celsius degrees)	Period
Pre-denaturation	95	3min.
Denaturation	95	30sec.
Annealing	55	30sec.
Elongation	72	3min.
Repetition-Denaturation,Annealing,Elongation	/	30times
Elongation	72	10min.

Procedure VI-electrophoresis

A.Add samples to the well comb: two samples of 10μl marker and 13μl of the samples in each group.

B.Connect to 160V electricity for approximately 20 minutes.

C.Examine the result with the illumination of ultraviolet radiation.

IPTG induced protein expression 、 purification and identification

IPTG induced protein expression

Materials

Reagent	Volume/mass
Agar solution	20ml
IPTG Mix	50ml
Bacterial fluid solution	20ml

Procedure

a)Expand/transfer the incubation container to 50mL centrifugal tube, add 20mL LB agar solution

b)Add 20mL bacterial fluid solution to the tube

c)Add 50mL IPTG to the tube and incubate at 16 degrees Celsius for 12 to 16 hours

The amount of IPTG added is based on the equation C1V1 = C2V2

-the ratio of concentration of IPTG to bacterial fluid solution is 1:1000

-in order to find the most suitable concentration of IPTG, we tested a variety of different concentrations based on amount of IPTG: 0.05mmol, 0.25mmol, 0.5mmol, 0.8mmol, 1mmolwhen OD600=0.4-0.8

d ) 16 degrees, overnight culture 12-16 hours.

Protein Purification

2.1 Ultrasonic crushing of cell membranes

*Essence: with ultrasonic waves, the membrane structures of bacteria BL21 are damaged and split. The electrical energy transfers to sound energy during this process, and the high-frequency sound waves in water can severely shock the bacteria.

*Other methods like alternating pH(e.g. alkaline splitting) present similar outcome. However, they possess lower efficiency.

Materials

Bacteria solution of BL21,Buffer HisA,Buffer HisB

Procedure

A. Obtain the 50ml centrifuge tubes containing cultured BL21 bacteria. Centrifuge it with the centrifuging force of 12000xg, the temperature of 4 Celsius Degree, and the time of approximately 10 minutes.

B. After centrifuging, discard the supernatant and retain the precipitate.

*The ppt. contains proteins required and the bacteria BL21.

C. Use pipette to measure 200μl of buffer HisA and add it to the centrifuge tube. Use the pipette to inject and retrieve the mixture in the tube continuously to float the precipitate (bacteria)in the solution.

D. Place the tube on the ice for ultrasonication with the lid uncovered. The process includes repetition of breaking for 3 seconds and stop for 3 seconds. The entire process lasts for about 15 minutes.

2.2 Purify the proteins

*Essence: during the design of the product vector, the DNA sections that form target protein all contain His group. In the proteins formed, His group can attach to the Nicole cations in Ni-NTA resin while other proteins can’t. The target enzymes and other proteins are thus separated.

*The result is also used for determining the optimum concentration of IPTG in induction.

Materials

Ni-NTA resin,His-A buffer,His-B buffer

Procedure

A. Obtain the product of ultrasonication, which is in a 50ml centrifuge tube. Centrifuge the product with centrifugal force of 8000xg and time of 5 minutes.

B. Obtain a 15ml centrifuge tube, a 50ml centrifuge tube ,and a 2ml centrifuge tube.

C. add all the supernatant from the centrifuging process to the 50ml centrifuge tube.

D. Use pipette to measure 200μl of Ni-NTA risen into the gravity column. Attach a 2ml centrifuge tube under the gravity column.

E. Use pipette to measure 100μl of His-A buffer into the gravity column to wash and filter the mixture in the gravity column. Discard the filtrate collected in the 2ml centrifuge tube.

F. Use pipette to add 100μl ddwater into the gravity column, wash and filter the mixture. Discard the mixture acquired in the 2ml centrifuge tube.

G. Add 5ml of supernatant into the gravity column. Place the tube for filtering.

H. add 3ml of His-A into the gravity column. Discard the filtrate.

*purpose: to wash the unwanted proteins out of the mixture

I. Add 3ml of His-B into the gravity column. Retain the filtrate in the centrifuge tube.

SDS-PAGE

*the gel required for the SDS-PAGE contains two sections: stacking gel and separating gel. The former aggregates protein molecules together, and the latter separates molecules like common electrophoresis.

*The reagents include β-mercaptoethanol, which breaks the disulfide bonds and eliminate the tertiary structure for the enzymes, and the SDS, which breaks the hydrogen bonds in the protein structure.

*The electrophoresis instrument requires specific procedures for operation

Materials

Reagent	Volume/mass
* Separating gel:	μL
2x Separating solution(10%)
Separating buffer
Coagulant
* Stacking gel:
2x stained stacking solution
Stacking buffer
ddwater	1000μL

Procedure

A. Pick the base and set it on a horizontal table. Let the side with a light-red stripe face to the person. Attach the gel model to the base.

B. Obtain the glass plates. Attach it to the model and ensure that the higher glass plate is placed toward the operator.

C. Examine that the model for gel is sealed. Use pipette to measure 1000μl ddwater and add it from the aperture between the two glass plates. Place the instrument at rest and observe whether water leaks from the bottom of the water or not. If the water doesn’t penetrate through the base, the model is sealed.

For separating gel:

A.Pick a plastic cup. Use pipette to measure 2.7ml of separating buffer and add it to the container.

B.Use pipette to measure 2.7ml of (2x) 10% separating solution and transfer it to the container.

C.Use pipette to measure 55μl of coagulant and add it to the container.

D.Stir the mixture to separate the three reagents evenly.

E.Quickly transfer the mixture to the electrophoresis instrument by using pipette. When the edge of the liquid observed has reached the while line indicated on the larger glass plate, stop adding the mixture.

*The mixture must be added quickly: the added sample will coagulate rapidly if the remaining samples are added slowly.

F.Use pipette to measure 1000μl of ddwater and add it to the model of electrophoresis.

G.Place the instrument at rest. Observe if the mixture has penetrated the base and leaked out. If this circumstance happens, operate the entire procedure again.

For stacking gel:

A.Pick a plastic cup. Use pipette to measure 750μl of (2x) stained stacking solution and add it to the container.

B.Use pipette to measure 750μl of stacking buffer and add it to the container.

C.Use pipette to measure 15μl of coagulant and add it to the container.

D.Stir the mixture.

E.Use pipette to transfer the mixture in the cup to the electrophoresis instrument by adding the mixture into the aperture between two glass plates. Continue to add the mixture until the model is full .

F.Place the instrument at rest until the stacking gel and separating gel have all coagulated.

Enzyme activity determination

1. Prepare for creating the standard curve

*Standard Curve is significant since it can transfer the result of absorbance(which reflects chromatic traits) to the concentration of uric acid remaining in the mixture, indicating that it can directly infer the efficiency of UAO from the indices acquired.

*Essence:

The difference in the concentration of Uric acid is directly related to the amount of Uric acid that has been digested by UAO, which is further related to the H2O2 formed and the absorbance of the products. The proportionality between Uric acid concentration and absorbance can thus be determined and a linear function can be deduced.

Materials:

Standard Uric acid solution, UAO and UAA sample

Procedure:

Ensure that the experiment need to create specified samples that will show a linear relationship between the absorbance(Final examination result of the decomposition for uric acid) and the concentration of the uric acid. The volume required for each sample is 250μl(Uric acid and water)+750μl(testing agent).
The volume for each group are thus as follow:

Sample\conc. of acid(μmol/ml)	0.25	0.125	0.0625	0.03125	0.015625	0.0073125	0
Standard Uric acid solution(μl)	25	12.5	6.25	3.13	1.56	0.73	0
Ddwater(μl)	225	237.5	243.75	246.87	248.44	249.27	250
Testing agent(μl)	750	750	750	750	750	750	750

Pix 6 1.5ml centrifuge tubes.
Use pipette to measure the standard uric acid solution required for each group and add them to the centrifuge tubes. The volumes are corresponded to the graph shown.
Use pipette to measure the ddwater required for each group and add them to the centrifuge tubes. The volumes are corresponded to the chart shown above.
Use pipette to measure 750μl testing agent with UAO,UAA and add it to the centrifuge tube in group 1. Operate the same procedure to the other 6 tubes.
Put the 7 centrifuge tubes in the 37 Celsius Degree environment for 30 minutes.
After reacting, examine the absorbance of the products by using ultraviolet spectrophotometer. Operate the measurement procedure for 5 times and calculate the average value.

Construct the system of testing

* Each group creates 6 testing groups with different concentrations of UAO and UAA.

* Essence:

The route of decomposing uric acid by UAO is:

Uric acid(+UAO)allantoin+H ₂ O ₂ +CO ₂

The examination focuses on testing the H ₂ O ₂ produced. If the amount of the product acquired in the testing of H ₂ O ₂ is high, the amount of H ₂ O ₂ formed in the decomposition reaction is high; it can thus be confirmed that the enzymatic activity for the UAO sample is high.

*H ₂ O ₂ possesses reducibility. The reagents used to test H ₂ O ₂ contains Fe ²⁺ cations. These ions are oxidized during the reaction and are transferred to Fe ³⁺ cations, which forms red brown quinone when combining with 4-Aminoantiprymine and phenol. The color is used for the chromatic examination, which determines the activity of UAO.

Materials

Reagent	Volume/mass
Ddwater	/
Standard Uric acid solution (conc. = 2.5μmol/ml)	50μL
UAO sample (The supernatant of the mixture in the centrifuging and ultrasonication yesterday is used, since the extracted sample possesses low concentration)	/
The testing agents (Contains UAO, 4-Aminoantiprymine, and Potassium ferrocyanide oxide ).	750μL

Procedure

A. pre-Information:

*Ensure the concentration of Uric acid required is 0.25 μmol/ml, the volume of the uric acid sample needed is 100μl, and the total volume for the sample is 300μl(enzyme+uric acid sample)+750μl(testing agents)

*Ensure that the concentration of UAO needs to be altered, and the amount of uric acid needs to be constant.

*The volumes of reagents required in different groups thus are:

Reagents\group	1	2	3	4	5	6(control group)
Uric acid sample( 0.25 μmol/ml)	100	100	100	100	100	0
UAO sample （ μl ）	0	50	100	150	200	300
Testing reagents(μl)	750	750	750	750	750	750
Ddwater	200	150	100	50	0	0

B. Pick six 1.5ml centrifuge tubes and label them 1~6.

C. Pick one 2ml centrifuge tube(the tube is used for preparing the uric acid sample). *Since the total volume required for uric acid sample is 500μl and the concentration of uric acid is 0.25μmol/ml, the ratio between standard uric acid sample(conc. : 2.5μl) and the ddwater is 1 : 9.

Use pipette to measure 50μl of standard uric acid sample and add it to the 2ml centrifuge tube. Use pipette to measure 450μl of ddwater and add it to the 2ml centrifuge tube. Attach the tube to the vortex.

D. Use pipette to measure 50μl, 100μl, 150μl, 200μl, and 300μl of UAO sample(supernatant) and add them correspondingly to the 1.5ml centrifuge tubes labelled 2,3,4, and 5. Attach the tubes to the vortex and directly place them on ice.

E. use pipette to measure 750μl of testing agents and add it to the centrifuge tube labelled 1. Repeat the procedure to other 5 tubes as well.

F. After adding testing agents, place the centrifuge tubes on ice for approximately 10 minutes, and then transfer them to the 37 Celsius Degree environment for 30 minutes.

*This is the procedure for the UAO to decompose the Uric acid.

G. After 30 minutes, use pipette to measure 200μl, 150μl, 100μl, and 50μl of ddwater and add them correspondingly to the centrifuge tubes labelled 1,2,3,and 4. Attach all 6 centrifuge tubes to the vortex.

*This procedure must be operated after the uric acid has been fully digested by UAO. If it is added earlier, the concentrations of the enzyme in different groups will be affected.

H. Add the samples to the Coated Wells: 3 groups refer to 3 lines labelled B,C, and D.

I. examine the absorbance of the products by using ultraviolet spectrophotometer. Operate the measurement procedure for 5 times and calculate the average value.

*Absorbance reflects the amount of the uric acids that have been decomposed by UAO.

Transformation 1917

1 . Transformation

Materials

Apparatus	Amount
Pipette (range:100-1000μL)	1
Centrifuge	1
Pipette (range:2-20μL)	1
Incubator	1
Rod	2
Culture plate	2

Procedure

A.Add the Nissle 1917 bacteria solution to the samples of vectors pGex-UAA and pGex-UAO. Flick the tube lightly.

B.Placed the sample on ice for 20 minutes.

C.Heat at 42 degrees Celsius for 45 seconds, then immediately put on ice for 2-3 minutes.

D.Add 900μL LB Agar solution, incubate for 30 minutes

E.Centrifuge at 5000xg for 4 minutes, extract 900μL of supernatant then discard

F.Continuously inject and retrieve the mixture in the centrifuge tube by using a pipette to float the bacteria in the centrifuge tube.

G.Use pipette to measure and apply 10μl product to agar plates, then use a rod to spread the bacteria solution evenly on the plate.

H.Use tapes to seal the culture plate. Incubate the plate for 12-16hr at 37 degrees Celsius. (The plates are placed upside down)

2. Isolated colonies validation

A. PCR system ：

Uric acid oxidase ( 981 bp)	Volume (ul)
2mix	10
Uric acid oxidase-R1	1
Uric acid oxidaseF1	1
pGEX-4T-1-UAO	1
ddH2O	7
Total	20

PCR procedure ：

procedure	temperature （℃）	Time
1	95	2 min
2	95	30 sec
3	55	30 sec
4	72	2 min
5	72	10 min
6	4	forever

A garose gel electrophoresis

1.Preparation of agarose gel : 100 mL 1 × TAE buffer was added to a conical flask, and 1 g agarose was weighed and placed in a conical flask, heated in a microwave oven for 2 min to completely melt, and shaken well.5 μL of nucleic acid dye Gel Red was added to the agarose gel, which was 1 % agarose gel containing nucleic acid dye. Install the gel plate and insert the comb. Pour the liquid gel into the gel plate, 3-4 mm high, avoid bubbles, and solidify at room temperature. After complete solidification, the comb was pulled out, the solid gel was transferred to the electrophoresis tank, 1 × TAE buffer was added, and the gel was completely immersed.

2.Spotting : each sample was added to the spotting hole of the gel in turn, and finally 10μL DNA Maker was added ( the gel around the sample hole should be prevented from being damaged and penetrating the bottom of the gel ) ;

3.Electrophoresis : cover the electrophoresis tank, the sample hole is located in the negative electrode of the electric field, turn on the power supply, 180V, 15min ;

4.Blue light observation : turn off the power supply, take out the gel, observe under the blue light glue instrument, the red fluorescent substance is DNA.

3.IPTG induction and fragmentation

When the bacteria were cultured to OD = 0.6, 0.5 mmol IPTG16 was added for overnight.

4. Ultrasonic crushing of cell membranes

*Essence: with ultrasonic waves, the membrane structures of bacteria Nissle 1917 are damaged and split. The electrical energy transfers to sound energy during this process, and the high-frequency sound waves in water can severely shock the bacteria.

*Other methods like alternating pH(e.g. alkaline splitting) present similar outcome. However, they possess lower efficiency.

Materials

Bacteria solution of Nissle 1917 ,Buffer HisA,Buffer HisB

Procedure

A. Obtain the 50ml centrifuge tubes containing cultured Nissle 1917 bacteria. Centrifuge it with the centrifuging force of 12000xg, the temperature of 4 Celsius Degree, and the time of approximately 10 minutes.

B. After centrifuging, discard the supernatant and retain the precipitate.

*The ppt. contains proteins required and the bacteria Nissle 1917 .