dsDNA were ordered from IDT. The dsDNA was amplified by PCR and cloned into standard iGEM backbone pSB1C3. Colony PCR was used to check the insertion of dsDNA. SDS-PAGE was used to check the overexpression of protein. The uricase activity of the biobricks was then validated by commercial uric acid kit.
Colony PCR confirms insertion of URI and HIU
The cloning of both uricase and HIU hydrolase are successful. Cloning PCR revealed correct number of base pairs of both uricase and HIU hydrolase. A 1090bp band and a 538bp band could be observed, which are uricase and HIU hydrolase repectivily.
SDS PAGE confirms the translation of uricase
Protein expression of both uricase and HIU hydrolase were also successful.
A single colony of BBa_K3058000 and BBa_K3058001 were picked to grow a 5mL overnight starter solution. Untranformed cells were used as control. After 1% inoculation, the cells were allowed to grow for 16 hours. The cells were then harvested and SDS-PAGE was conducted. When comparing to the control, a overexpressed band could be observed in uricase-expressing and HIU hydrolase-expressing E.coli. The location matches the theoretical size, which are 34.2kDa and 13.56kDa respectively. This indicated that our biobrick could express our expected protein.
Construction of uric-acid-inducible uricase generator
(BBa_K3058002) using Gibson Assembly
Initially, we could not synthesize a biobrick(BBa_K3058002) because it was longer than 3000bp. Luckily, through collaboration, Hong_Kong_JSS 2019 team helped us to synthesize the DNA using Gibson assembly. The expected size was correctly synthesized. The validation of the biobrick is still in progress.
Endogenous uric acid transporter may allow cellular uptake of uric acid.
To check if E.coli could intake uric acid, we mix E.coli with uric acid-containing limited-medium and obtained the change in extracellular and intracellular uric acid concentration. At the beginning, 100% of the uric acid was located in the supernatant but not in the cell pellet after centrifugation. After 2 days, arround 50% of the uric acid is translocated into the cell pellet. This indicated that our intracellular approach is possible.
Experimental Protocols
Set up PCR mixture as follow :
Component
Volume
Green Master Mix
25 μl
Primer Mix (10 μM)
2 μl
Insert
1 μl
Milli Q
22 μl
Mix reagents in tubes by pipetting the solution up and down slowly.
Quick spin the PCR tube to ensure the mixture is in the bottom of the tube.
Put it in the thermocycler (PCR machine) and set the cycle information.
Start the cycle and wait till it is finished.
Prepare your 1% gel by using 0.5g of agarose in 50 ml of TAE buffer.
Add 2.5ul of Ethidium Bromide before you pour your gel into the chamber.
Mix 5ul of DNA with 1ul of loading buffer by pipetting up and down a couple of times.
Load your samples and appropriate marker into your wells
Incubate at 37°C and 250 rpm for 60-120 minutes.
Apply 130 volts to the chamber for 20 mins.
Check your gel using a transilluminator or other UV emitting device.
Re-suspend cells by light vortexing
Plate resuspended cells as above
Incubate overnight at 37°C with plates upside down.
Trimp the DNA fragment from the agarose gel and transfer the gel to 1.5 mL
microfuge tube followed by weighing the gel slice.
4 volumes of gel dissolving buffer is transferred to the gel slice.
The sample is incubated between 37 o C – 55 o C and vortex the sample
periodically until the gel slice is completely dissolved.
Insert the column into collection tube and the sample is loaded onto the
column. The column is the spin for 1 minute, the discard the flow-through.
The column is re-inserted into collection tube. 200 μL DNA Wash Buffer and
spin for 1 minute and repeat this step.
The column is transferred to a clean 1.5 mL microfuge tube.
6 μL DNA elution buffer is added to the center of the matrix. After 1 minute,
spin the sample to elute DNA.
Prepare the PSB1C3 plasmid:
Component
Volume
Buffer
5 μl
EcoRI
0.5 μl
PST1
0.5 μl
PSB1C3
4 μl
Milli Q
40 μl
Prepare the Insert:
Component
Volume
Buffer
5 μl
EcoRI
0.5 μl
PST1
0.5 μl
Insert
20 μl
Milli Q
24 μl
Pipette the solution up and down to ensure all reagents are mixed well.
Place the reaction mixture at 37 oC incubation or dry bath for 2-4 hours.
Purify the DNA by PCR purification kit/gel extraction kit for downstream process.
Mix the solution mixture according to the contents below:
Prepare the Insert:
Component
Volume
Buffer
4 μl
Plasmid
3 μl
Insert
10 μl
Ligase
1 μl
Milli Q
2 μl
Prepare the Control:
Component
Volume
Buffer
4 μl
Plasmid
3 μl
Insert
0 μl
Ligase
1 μl
Milli Q
12 μl
The solution mixtures is then incubated in 4 o C for 16 hours
Prepare the Ca/glycerol buffer as follow and flow through 0.22 μM filter.
Composition
Volume
0.6 M CaCl2
10 ml
0.5 M PIPES pH7.0
2 ml
Glycerol
15 ml
H2O
73 ml
Total
100 ml
Spread the bacterial cell (DH5α, BL21 and Nissle 1917) for plasmid replication on the LB.
Pick single colony in 5 ml LB culture medium and grow overnight at 37 °C by shaking at ~220 rpm.
Add 1ml overnight culture each to two of 100 ml flasks and grow the cell culture to achieve OD 600 = 0.25-0.4 (~ 3 h)
Transfer the cell culture (total 200 ml) to four of 50 ml sterile centrifugation tubes.
Collect the cell by centrifuging at 1000 g for 10 min at 4°C.
Gently resuspend the cell in each tube with 10 ml ice-old Ca/glycerol buffer. Keep the solution ice-cold. * Cells must remain clod for the rest of the procedures!
Collect the cell by centrifuging at 1000 g for 10 min at 4 °C.
Centrifuge at 13,000 rpm for 1 min.
Gently resuspend the cell in each tube with 1.25 ml ice-old Ca/glycerol buffer.
Centrifuge at 13,000 rpm for 1 min.
Column dring with centrifuge 13.000rpm for 1 min.
Transfer all cells to one tube.
Dispense 100 μl aliquots of competent cells into each Eppendorf.
Store at -80 °C.
Test the transformation efficiency of competent cells with antibiotics resistance plamids at different concentrations.
Pre-heat the water to 42 o C in water bath and warm the LB solution in incubator
at 37 o C.
Obtain -80 o C DH5α and remove from ice bath, to allow it to liquefy.
Add DNA in a ratio of 100 µL DH5α to 20 µL of DNA in the Eppendorf.
Wait 10-20 minutes in Ice Bath in order to allow DNA to enclose the cell
membrane of DH5α.
Immerse the Eppendorf containing the mixture into water bath of 42 o C for 1
min exactly as a heat shock procedure. The Eppendorf is then taken to an ice
bath for 1-2 minutes to allow the pores on the cell membrane to close up.
Add 1 mL of LB solution at 37 o C to the Eppendorf, the mixture will then be
incubated at 37 DC for 1.5 hours.
Centrifuge the Eppendorf for 1 minute 25 o C at 13,000 rpm.
Remove excess liquids and remain 100 µL of liquid in the centrifuge tube.
Pipette up and down to wash the pellet.
Use a pipette to extract 100 µL of the liquid then transfer and spread the liquid
on the agar plate.
5 mL with 5 μL chloramphenicol of LB solution is placed inside 15 mL falcon
A colony is picked from the cultured plate after transformation and
transferred to the falcon containing the solution under fire.
Loosen the cover of the flacon.
Incubate the solution in the flacon at 37 o C for a day..
Dilute the well mixed solution containing the bacteria and LB solution to 0.5
A of OD600 from UV spectrophotometer.
500 μL of sample is collected and centrifuged at 13000 r.p.m. in a micro-
centrifuge tube.
Discard the LB solution.
Re-suspend in 50 μL of Milli Q water and transfer further 50 μL of laminin
sample buffer.
Incubate the solution mixture in PCR at 95 o C for 15 minutes.
Run the SDS page using precision plus protein kaleidoscope standards ladder
Prepare the following reaction mixture as the table shown below
Component
Volume
Amount of DNA fragment
3.45 μL of fragment 1 + 3.57 μL of fragment 2
NEBuilder HiFi DNA
Assembly Master Mix
10 μL
Deionized Water
2 μL
Total volume
10 μl
Milli Q
2 μl
The reaction mixture is then incubated in a thermocycler at 50 o C for 15
minutes.
After incubation, PCR is carried out.
Pipette 50 μL of the samples into separate wells of a microplate.
Begin the reactions by adding 50 μL of the Amplex® Red reagent.
Incubate the reactions for 30 minutes or longer at 37°C, protected from light. The absorbance may be measured at multiple time points to follow the kinetics of the reactions.
Measure the absorbance in a microplate reader using excitation with absorbance at ~560 nm.
