Experiments | Keystone

Extracted DNA PCR System

Prepare the PCR reaction system added according to the proportion below. Refer to different tables depending on the enzyme type used.

For 2 X Taq Plus PCR Master Mix, 20 ul* PCR reaction system

Reagent	Amounts (ul)
2 X Taq Plus PCR Master Mix	10
ddH2O	6
Sample DNA template	2
Forward Primer	1
Reverse Primer	1

For Lamp Master Mix, 20 ul* PCR reaction system

Reagent	Amounts (ul)
Lamp Master Mix	10
ddH2O	7.4
Sample DNA template	1
Forward Primer	0.8
Reverse Primer	0.8

For Phusion Pfu DNA Polymerase mix, 20 ul* PCR reaction system

Reagent	Amounts (ul)
ddH2O	6
Pfu Buffer	8
Sample DNA template	2
Forward Primer	1.6
Reverse Primer	1.6
Pfu Polymerase	0.8

*For other amounts (ex. A 50 ul reaction system) multiply the amounts by a common factor.

Once completed, jump to the thermocycler protocol to edit the program in the thermocycler.

Bacterial & Cyanobacterial Colonies PCR System

To prepare the DNA template, you need to first extract DNA from a single colony. To do this, follow these instructions:

*Note: When picking the single colony, avoid selecting colonies in densely distributed areas as to avoid accidentally touching other colonies with the tip of your micropipette.

Next, add in the reagents into an empty PCR tube. Depending on the enzyme type used, refer to the tables below:

For 2 X Taq Plus PCR Master Mix, 20 ul* PCR reaction system

Reagent	Amounts (ul)
2 X Taq Plus PCR Master Mix	10
ddH2O	6
Sample DNA template	2
Forward Primer	1
Reverse Primer	1

For Lamp Master Mix, 20 ul* PCR reaction system

Reagent	Amounts (ul)
Lamp Master Mix	10
ddH2O	7.4
Sample DNA template	1
Forward Primer	0.8
Reverse Primer	0.8

For Phusion Pfu DNA Polymerase mix, 20 ul* PCR reaction system

Reagent	Amounts (ul)
ddH2O	6
Pfu Buffer	8
Sample DNA template	2
Forward Primer	1.6
Reverse Primer	1.6
Pfu Polymerase	0.8

*Again, for other reaction system sizes (50 ul etc.), just multiply everything by a common factor.

Thermocycler PCR

Place the PCR tube in the thermocycler and start the program below.

Note depending on the enzyme used there may be slight variation, the one below is only an example of what the protocol could be. Check the notebook for more information on the specific thermocycler protocols for each step of our experiment.

Thermocycler PCR Protocol

Step	Degree(C)	Time
1	94	10 min
2	94	30sec
3	Tm-5	30sec
4	72	Xs
5	Go back to step 2 and complete 30-35 cycles.
6	72	5min
7	10	Ifinity

*Tm means melting temperature of the primers used

*Due to the general rule of the amplification efficiency of the enzyme as 1kb/min amplicon, the x value is equal to the length (kb) of the DNA fragment amplified by PCR

After finishing the entire PCR process, store the PCR product in a 4 C Fridge

Agarose Gel Electrophoresis

According to the amount of TAE buffer needed, mix the 1% of agarose powder with 1X TAE buffer.
Heat up the solution until it turns clear.
When the solution is cooled to around 60 degrees Celsius, add the DNA dye into the solution.
Pour the solution slowly into the gel chamber and put in the comb after making sure that the casting tray will not leak.
After the gel solidifies, remove the combs.
Use the pipette, insert the DNA marker into the first well and slight amounts of the samples into each well starting from the second well.
Run the gel in the voltage and time according to the need of the sample inserted.
Place the gel on the gel imaging system and view the results.

Agarose Gel DNA Recycling

Cut the target DNA strip from the agarose gel into a centrifuge tube and weigh it.
Add three times the volume of sol solution, 50 degrees Celsius of water bath for 10 minutes until the gel completely dissolves to liquid.
Add the gel liquid to spin column CA1 or CA2 and the column to the collection tube, centrifuge at 13,000 rpm for 30 seconds, pour out the waste liquid.
Add 700 uL rinse liquid PW, centrifuge at 13,000 rpm for 30 seconds, pour out the waste liquid.
Add 500 uL rinse liquid PW, centrifuge at 13,000 rpm for 30 seconds, pour the waste liquid. Centrifuge at 13,000 rpm for 2 minutes, then put the spin columns in room temperature until it dries.
Put the spin column into a clean centrifuge tube, elution buffer EB to it, centrifuge at 13,000 rpm for a minute and collect the DNQA solution.
Step 6 could be repeated to increase the recycled amount of DNA.

SDS-PAGE Gel Electrophoresis

Preparation of Gel:

Mix all reagents to prepare the gel, TEMED added at last.
Pour the gel into pouring chamber.
Add butanol to remove unwanted air bubbles
Insert the comb into the space between the glass plates.

Sample Preparation

Add pure water to the beaker.
Add 2-mercaptoethanol to the sample buffer.
Place the buffer solution in a microcentrifuge tube and add the protein sample to it.
Place the MW marker in a separate tube.
Boil the sample for less than 5 minutes to completely denature the protein.

Electrophoresis

Remove the gel cassette from the casting table and place it in the electrode assembly.
Secure the electrode assembly to the fixture rack.
Pour the 1x Electrophoresis Buffer into the opening of the casting frame to fill the gel wells.
Aspirate 30ml of denaturing sample into the wells.
Cover the tank with a lid and connect the device to the power supply.
Run the sample at 30mA for about 1 hour and see the bands under UV light.

CA Enzyme activity

Ingredients

Reagent name	Specification	Preservation condition
Extracting solution	Liquid 60mL x 1 bottle	2-8˚C
Reagent 1	Liquid 50mL x 1 bottle	2-8˚C
Reagent 2	Powder x 2 bottles	2-8˚C
Standard substance	Liquid x 1	2-8˚C

Carbonic Anhydrase (CA, EC4.2.1.1) is a metallic enzyme with Zn2+ as the active center, which can be used to efficiently catalyze reversible hydration reaction of carbon dioxide: CO2+H2O⇋HCO3-+H+, with a catalytic rate up to 107 times that of natural conditions. It is one of the fastest catalytic enzymes known.

Carbonic anhydrase can catalyze the reaction of acetic acid to nitrophenyl ester to produce p-nitrophenol. The activity of carbonic anhydrase is reflected by detecting the increasing rate of absorbance value at 405nm.

Protocols

Sample processing:

Tissue: According to tissue quality (g): extraction liquid volume (mL) is 1:5~10 (it is recommended to weigh about 0.1g tissue and add 1mL extraction liquid), ice bath homogenization is carried out. Centrifuge 8000g at 4°C for 10min, take the supernatant and put it on the ice to be measured.
Bacteria or cultured cells: Collect bacteria or cells into the centrifuge tube first, and discard the supernatant after centrifugation; According to the number of bacteria or cells (104): the extraction liquid volume (mL) is 500~1000:1 ratio (it is recommended that 5 million bacteria or cells be added to 1mL of extraction liquid), ultrasonic crushing bacteria or cells (ice bath, power 200W, ultrasonic 3s, interval 10s, repeat 30 times), 8000g, Centrifuge at 4°C for 10min, take the supernatant and put it on the ice to be measured.
Liquid: direct measurement. (If the solution is cloudy, centrifuge the supernatant and then determine).

Measuring procedure

1. Preheat visible spectrophotometer more than 30min, adjust the wavelength to 405nm, distilled water zero.

2. Standard tube determination:

Standard tube determination: add 100μL standard liquid in the cuvette, 900μL reagent 1, fully mixed at 405nm after determination of light absorption value, recorded as A_standard.
Determination of standard blank tube: 100μL distilled water and 900μL reagent were added to the colorimetric dish, and the absorption value was determined at 405nm after full mixing, and was recorded as A_standard blank.
Calculate ∆A_standard = A_standard - A_standard blank. (Standard tubes and standard blank tubes only need 1-2 times.)

Operation table

Reagent	Measuring tube (μL)	Blank tube (μL)
Sample	100	-
Extract	-	100
Reagent 1	700	700
Reagent 2 working fluid	200	200

The above reagents were added into 1mL glass cuvettes successively according to the sample adding table, immediately and thoroughly mixed to determine the light absorption value A1 at 10s at 405nm, quickly placed in 37°C water bath or constant temperature incubator for 5min, and quickly wiped to determine the light absorption value A2 at 5min10s. Calculate A_measure = A2_- A1_measure, A_blank = A2_blank -A1_blank, ∆A = A_determination - A_blank

Protein Concentration Measurement

In our experiments, protein concentration is measured using the Enhanced BCA Protein Assay Kit. A brief overview of the procedures in this kit is provided below.

Add 0, 1, 2, 4, 8, 12, 16, and 20 μL protein standard into the standard wells on a 96 well plate, respectively. Add protein standard diluent to make up to 20 μL in each well. In other words, the concentration of protein standard in each standard well will be 0, 0.025, 0.05, 0.1, 0.2, 0.3, 0.4, and 0.5 mg/mL, respectively.
Add an appropriate volume of sample to the sample wells of the 96 well plate. If the sample is less than 20 μL, add protein standard diluent to make up to 20 μL. Record the sample volume.
Add 200 μL BCA working solution into each well and place the 96 well plate at 37˚C for 20-30 minutes.
1. Note: the plate can also be placed at room temperature for 2 hours or at 60˚C for 30 minutes. When measuring protein concentration using the BCA method, the color of the mixture in each well will gradually deepen over time, and the color reaction will accelerate along with increases in temperature. If the sample concentration is low, it is better to incubate at a higher temperature or extend the incubation time appropriately.
Measure the absorbance at A562 or wavelength within the range of 540-595 nm, using a microplate reader.
Calculate the protein concentration of the sample based on the standard curve and the sample volume.

BioBrick Protocol

Mix 10g of alginate into 100ml of BG11, mix with a magnetic stirrer at 90°C.
After gelatin fully dissolves in the medium, add 0.1M NaHCO, into the solution until the pH value becomes 7.6
When the alginate solution cools to below 36˚C, add 20ml of cyanobacteria-containing liquid medium and mix with the alginate solution.
Add 40g of pre-treated sea sand (small grains) and river sand (large grains) mixture and mix well with the alginate and cyanobacteria mixture.
Note: treatment for aggregates procedure that follows:
1. Soak sand in 4% HCl solution for 24h
2. Take out the sand and rinse with tap water until the pH value becomes 7.0
3. Put the sand into the furnace and bake for 48h at 80˚C.
Pour the mixture into a square mould and flatten the top surface.
Pour a certain amount of CaCl2 2H2O solution (1M) onto the surface of the mixture until the surface is completely covered. By this point, the surface should be quickly solidified.
Gently lift the hydrogel and pour more CaCI2-2H2O solution (1M) onto the bottom of the mould. Wait for a few minutes, then lift the hydrogel and immerse it in 0.5M of CaCI2:2H2O solution overnight.
Then, dry the hydrogel in a dryer at 60°C for about 12 hours to obtain dried Biostone.

CA effect on Biostone Desiccation

Soak fine sea sand in 4% HCl solution for 24h
Take out the fine sea sand and rinse with tap water until the pH value becomes 7.0
Put the sea sand into the furnace and bake for 48h at 80˚C.
Repeate steps 1-4 for coarse river sand
50mL 5% sodium alginate gel using BG11 medium as the solvent. Put the processed sand into two square molds and then pour in the sodium alginate gel solution. Fully homogenize them afterwards.
One square mold adds purified aqueous CA and the other one without CA. The aqueous CA was added after the homogenization of sodium alginate solution and aggregates.
0.1M (~1.0%) CaCl2·2H2O solution was added to both two BioStones by spraying at the end.

Carbonic Anhydrase Extraction Protocol

Preparing tissue samples, thaw them on ice before starting the extraction.
Place samples in a pre-chilled homogenization buffer.
Centrifuge the homogenate at a low speed for 10-15 minutes at 4°C
Collect the supernatant carefully and transfer it to a microcentrifuge tube
Measure the protein concentration in the supernatant using a protein quantification method
Aliquot the supernatant into smaller portions and store them at -80°C

Cyanobacteria Transformation Protocol

Prepare a healthy culture of cyanobacteria in the exponential growth phase.
Isolate or obtain the plasmid DNA containing the target gene. Verify its purity and concentration.
Harvest cyanobacterial cells by centrifugation at a low speed and wash them with fresh cyanobacterial growth medium
Mix the harvested cyanobacterial cells with the purified plasmid DNA. Adjust the DNA concentration based on cyanobacterial strain and transformation efficiency.
Load the cell-DNA mixture into a cuvette or transformation chamber. Use an electroporator following set the voltage, resistance, and capacitance parameters
Transfer the electroporated cells into a fresh, pre-warmed cyanobacterial growth medium.
Incubate cells under suitable conditions
Add the appropriate antibiotic to the recovery medium to select for transformed cells
Plate the transformed cells onto agar plates
Incubate the plates under the same controlled conditions
Analyze resulting colonies for the presence of the target gene using techniques such as PCR and sequencing

E.Coli Transformation Protocol

Prepare a fresh, frozen E. coli culture onto an LB agar plate. Incubate the plate at 37°C overnight.
Isolate or obtain the plasmid DNA containing the target gene. Verify its purity and concentration.
Take a tube of competent E. coli cells from the -80°C freezer and thaw it on ice.
Add 1-5 μl of the plasmid DNA to the thawed competent cells.
Incubate the cell-DNA mixture on ice for 30 minutes.
Heat shock the mixture by placing it in a 42°C water bath for 45 seconds
Transfer the tube back to ice and let it cool for 2 minutes.
Add 950 μl of pre-warmed SOC medium to the cell-DNA mixture.
Incubate the tube at 37°C in a shaking incubator for 1-2 hours
Plate a portion of the transformed cell suspension onto an LB agar plate containing the appropriate antibiotic. Spread the cells evenly using a sterile spreader.
Incubate the plates inverted at 37°C overnight or until colonies are visible.
Select colonies on the LB agar plate with antibiotic.
Verify the presence of the target gene by performing plasmid isolation and techniques such as PCR or DNA sequencing.

Gibson Assembly

Protocol for Gibson Assembly

Set reaction on ice
Incubate samples in a thermocycler at 50°C for 15 minutes when 2 or 3 fragments are being assembled or 60 minutes when 4-6 fragments are being assembled. Following incubation, store samples on ice or at –20°C for subsequent transformation.
1. Note: Extended incubation up to 60 minutes may help to improve assembly efficiency in some cases
Transform NEB 5-alpha Competent E. coli cells (provided with the kit) with 2 μl of the assembly reaction, following the transformation protocol.

Golden Gate Assembly

Protocol for Golden Gate Assembly

Set up the following reaction mix on ice:
1. X µl DNA Insert PCR reactions (150 ng of DNA each or 2:1 molar ratio, insert:plasmid)
2. 1 µl (~75 ng) Vector/Plasmid/Backbone
  1. Or Golden Gate kit pGGA destination plasmid
3. 1 µl Golden Assembly Master Mix
  1. Use 2 µl for assemblies of > 10 inserts
4. If you're using enzymes instead of master mix:
  1. Insert + Plasmid (2:1 molar ratio)
  2. 2.5 µl T4 DNA Ligase Buffer (extremely heat sensitive, will not survive multiple heat/thaw cycles)
  3. 0.5 µl T4 DNA Ligase (extremely heat sensitive)
  4. 1.5 µl BsaI-HFv2 (relatively heat sensitive)
  5. Make up to 20 µl of dH2O
5. 20 - X - 2 dH2O
Mix gently by pipetting up and down. Tap on the bench or briefly centrifuge (1 sec) to ensure no bubbles in the reaction mixture.
Transfer the reaction tube(s) to a thermal cycler and run the following program:
1. (5 min 37°C → 5 min 16°C) x 30 cycles
2. 5 min 60°C
3. If you're running overnight, add a Hold at 4°C. The next morning, briefly heat the reaction to 60°C before proceeding.
Add 2 µl assembly reaction to 50 µl thawed chemically competent cells and allow to sit on ice for 15-30 minutes.
1. Make sure you include a positive and negative control (2 µl uncut plasmid, 10 µl dH2O)
Take your esky of ice over to the 42°C waterbath or 42°C heat block. Put tubes in a floatie (or) hold in the water bath (or) push tubes into the slots of the heat block. Allow 45 seconds for heat shock. (Plus or minus 10 seconds, this needs to be exact!). Then transfer the cells straight back onto ice (embed into ice, as above, don’t just rest on top).
Allow transformation mixtures to sit for 2 min on ice, then add 1 ml sterile LB broth to each tube. You can also use more fancy media (e.g. SOC or SOB), but there is not that much difference.
Incubate on 37°C shaker for 1 hour. Put the tubes horizontal so they get good shaking action. eg. put the tubes laying flat on the shaker platform and masking-tape into place. Make sure the lids are tight! You can incubate without shaking, and you can incubate for less time (30 min), but it won’t work as well in these cases.
1. The role of this ‘recovery’ step is to allow the cells to create the proteins required for antibiotic resistance.
2. Recovery is not necessary for Ampicillin resistance plasmids. Proceed to plating.
Label the LB-antibiotic plates before starting the next bit; you need two plates for each ligation condition or plasmid type, since we will plate out two different cell concentrations of each to ensure we get countable/pickable numbers of colonies. Double check the plates to ensure you are using the correct type of antibiotic(s) for the type of plasmid(s) you are using.
Pipette 100 µl of the first cell suspension onto one LB-antibiotic plate (label ‘100 µl’ in addition to other info) and spread it over the plate using the glass rod (liquid > solid) Sterile Technique.
Spread 100 µl of the remaining samples, each onto a separate, appropriately-labelled plate.
Centrifuge all the tubes at ~15,000 rpm for 1 minute in a micro-centrifuge. Pour off most of the supernatant into culture waste (being careful not to touch the tubes on the edge of the culture waste bottle). Leave a little bit of liquid behind (about one or two drops).
1. The purpose of this spin and second plating is to ensure you avoid getting too few colonies or a confluent lawn when you check your plates tomorrow.
Vortex the cells in the remaining liquid for about 10 seconds, until they are not sticking to tube anymore, and you have a nice smooth, even, cell suspension.
Pipette the cells from the first cell suspension onto the appropriate pre-labelled LB-antibiotic plate (label with ‘pellet’ in addition to other info), spread plate as above. Repeat for the remaining samples and controls.
Incubate all plates at 37°C overnight. Note that for some plasmids and ligations, it may be beneficial to instead try room temp for 2-3 days – this lowers the copy number of pUC type plasmids, and is useful to allow retrieval of clones that might be toxic to the host.

Overlap PCR Protocol

We used the lamp enzyme for this. There are two rounds of PCRs to achieve the overlap results.

For round 1:

For a 20 ul reaction system:

Reagent	Amounts (ul)
Lamp	10
DNA template A	1
DNA template B	1
Primer A F/R	0.8
Primer B F/R	0.8
ddH20	up to 20 ul*

*It’s written as up to 20 ul as depending on the number of DNA templates, the amount of ddH20 used may vary

Use gel electrophoresis to check if the results from the first round of PCR are what you want, if it is indeed what you want, purify the DNA out of the gel to prepare for the second round of PCR.

For round 2:

50 ul Reaction System:

Reagent	Amounts (ul)
Lamp	25
DNA template	2.5
Primer A F	2
Primer B R	2
ddH20	up to 50 ul*

Prepare this through the same series of steps as the ones written for round 1.

Use gel electrophoresis again and double check if the results are what you wanted. If so, purify the DNA out of the gel again and store for future use.