For a 50 ml mixture:
1.Make a fresh 1 M Sodium Bicarbonate solution in MilliQ water and make steril using a filter.
2.Add 500 µL of the Sodium Bicarbonate, 125 µL of each BG-11 stock: 1,2 and 3 and 43,7 ml
sterile MilliQ water to a 100 ml flask.
3.(optional) add antibiotics until a total concentration of 1:1000
1.Dissolve 1.5 gr Agar in 95 mL MilliQ water and autoclave 15 min at 121 degrees.
2.Cool to 55 ºC in stove
3.Add to the agar flask:
a.BG-11 stocks 1,2 and 3, stock 3 without EDTA 250 µL each
b.Thiosulfate 30 % 1 ml
c.Antibiotics 100 µL (optional)
d.Pipps 25 mM stock 6 ml
4.Pour plates
Stock 1
1.Dissolve CaCl2·2H2O in 700 mL MilliQ (MQ) water in a measuring cylinder.
2.Subsequently add NaNO3 and dissolve it under vigorous stirring.
3.Submerge the measuring cylinder in warm water.
4.When fully dissolved, cool to room temperature.
5.Adjusted to 1 L and filter sterilized.
Note :The solution should be colorless.
Stock 2
1.Dissolved EDTA Na2·2H2O in 400 mL MQ water in a plastic measuring cylinder
2.Add FeCl3·6H2O and left stirring overnight
3.MgSO4·7H2O is added to 150 mL MQ water in another measuring cylinder and stir until fully dissolved
4.Subsequently add this to the FeCl3·6H2O-EDTA Na2·2H2O solution together with 400 mL of stock 4, adjusted to 1 L and filter sterilized
Note: The solution is a greenish yellow and should not discolor the filter. If it does, the solution should be remade
Stock 3
1.The trace metals are added in the order they are listed in Table 1 to 1 L of MQ water
2.filter sterilized when fully dissolved.
Note: The solution should be colorless.
|
Stock solution
|
Compound
|
(g/L)
|
Lab Code
|
|
1
|
NaNO3
CaCl2·2H2O
|
600
14.4
|
N126
C13
|
|
2
|
MgSO4·7H2O
FeCl3·6H2O
EDTA Na2·2H2O
Trace Metal Mix
|
30.0
1.62
2.24
400 mL
|
M12
F5
E5
(Stock 4)
|
|
3
|
K2HPO4
EDTA Na2·2H2O
|
16.0
5.2
|
K70
E5
|
|
4
|
H3BO3
MnCl2·4H2O
ZnSO4·7H2O
Na2MoO4·2H2O
CuSO4·5H2O
Co(NO3)2·6H2O
|
2.86
1.81
0.222
0.391
0.079
0.049
|
B43
M52
Z18
N123
C202 / C204 C174
|
1. Dissolve the following components in MQ water 0.5 L
a. Yeast Extract 5 g
b. Tryptone 10 g
c. NaCl 10 g
2. Autoclave for 20 min at 121°C.
3. Optional: add antibiotics
a. Ampicillin final concentration100 μg. mL-1
b. Spectinomycin final concentration 50 μg mL-1
c. Kanamycin final concentration 50 μg mL-1
d. Chloramphenicol final concentration 34 mg mL-1
- Dissolve the following components in MQ water 0.5 L
- Yeast Extract 5 g
- Tryptone 10 g
- NaCl 10 g
- Agar 15 g
- Autoclave for 20 min at 121°C.
- Optional: add antibiotics
- Ampicillin final concentration100 μg. mL-1
- Spectinomycin final concentration 50 μg. mL-1
- Kanamycin final concentration 50 μg. mL-1
- Chloramphenicol final concentration 34 mg. mL-1
- Pick a single clone and inoculate into the liquid LB media, with antibiotics if necessary. Incubate overnight at 37°C, while shaking at 250 rpm.
- For 1 ml glycerol stock, add 800 µL cultures and mixed with 200 μL sterilized 80% glycerol. Gently mix, and put into the -80°C freezer.
- Prepare a relatively large amount of liquid culture at a OD around 1 to 2.5 (better if OD is lower than 3 in the flask) in BG-11 media.
- Add 800 μL cultures to 200 µL sterilized 80% glycerol.
- Gently mix and put into the -80°C freezer.
- Measure OD730 of preculture
- Calculate culture volume for OD730 0.1
- Divide culture volume by OD730 of preculture
- Subtract value from step 3. from total volume
- Subtract buffer (PIPPS-KOH at pH = 8.00) volume
- Calculate buffer (500mM) volume for a concentration of 25mM
- Subtract stocks volume
- Stocks are 400x concentrated (total volume/400)
- Multiply by three (stocks 1, 2 and 3 in equal concentrations)
- (Optional for experiments with NaHCO3) Subtract NaHCO3 solution volume
- Make a fresh 500mM NaHCO3 solution in MilliQ water
- Calculate NaHCO3 solution volume for desired concentration in total volume
- The value obtained is the MilliQ water volume to start preparing the master mix
- Add buffer volume from step 4.
- Add stocks as calculated in step 5.
- (Optional for experiments with NaHCO3) Add NaHCO3 solution volume using a sterile filter
- Add culture volume from step 2.
Basis medium
1. Add all compounds in the table below to reach the required amount per volume
|
Formula
|
Molecular Mass (g/mol)
|
Molarity (mol/L)
|
Mass (g/L)
|
|
Na2HPO4
|
141.96
|
0.0423
|
6
|
|
NaCl
|
58.44
|
0.0085
|
0.5
|
|
KH2PO4
|
136.086
|
0.022
|
3
|
|
NH4Cl
|
53.5
|
0.0187
|
1
|
|
MgSO4 · 7 H2O
|
246.47
|
0.001
|
0.25
|
|
CaCl2 · 2 H2O
|
147.1
|
0.0001
|
0.015
|
2. Add all compounds in the below to reach the required amount per volume (NOTE: Supplement for growth-related tests)
|
Compound
|
Molecular Mass (g/mol)
|
Molarity (mol/L)
|
Mass (g/L)
|
|
Glycerol
|
92.09
|
0.21718
|
20
|
|
L-Trp
|
204.23
|
0.00049
|
0.1
|
|
L-Phe
|
165.19
|
0.00061
|
0.1
|
|
L-Tyr
|
181.19
|
0.00055
|
0.1
|
3. Add Mili-Q to reach desired total volume
4. Mix solution well
5. Filter sterilize solution in a sterile flask
Preparation of M9 minimal medium agar stock solution without amino acids (Stock 1)
1. Add all compounds in the table below to reach the required amount per volume
|
Formula
|
Molecular Mass (g/mol)
|
Molarity (mol/L)
|
Mass (g/L)
|
|
Na2HPO4
|
141.96
|
0.0423
|
6
|
|
NaCl
|
58.44
|
0.0085
|
0.5
|
|
KH2PO4
|
136.086
|
0.0220
|
3
|
|
NH4Cl
|
53.5
|
0.0187
|
1
|
|
MgSO4 · 7 H2O
|
246.47
|
0.0010
|
0.25
|
|
CaCl2 · 2 H2O
|
147.1
|
0.0001
|
0.015
|
|
Glycerol
|
92.09
|
0.21718
|
20
|
|
Agar
|
336.33
|
0.0446
|
15
|
2. Mix compounds well using a magnetic stirrer
3. Autoclave for 20 minutes on 121 degrees Celcius
4. Store in stove at 55 degrees Celsius
Preparation of amino stock solution (Stock 2)
1. Add the following compounds (Table 2) to beaker
|
Compound
|
Molecular Mass (g/mol)
|
Molarity (mol/L)
|
Mass (g/L)
|
Solubility (g/L)
|
|
L-Trp
|
204.23
|
0.00049
|
0.1
|
11.4
|
|
L-Phe
|
165.19
|
0.00061
|
0.1
|
26.9
|
|
L-Tyr
|
181.19
|
0.00055
|
0.1
|
0.479
|
2. Mix compounds well using a magnetic stirrer
3. Filter sterilize compounds using a syringe and sterile filter
Final M9 minimal medium plates
- Add Stock 2 to Stock 1 in sterile flow hood to make final M9 minimal medium agar solution
- If necessary, add antibiotic supplements or other required supplements (e.g. pyruvate) to the plates for experiments
- Poor plates in flow hood and let set for about 10 minutes
- Close plates and store in sterile bag with name and date in cold chamber at 4 degrees Celsius
- PCR amplification of individual fragments
2x Phusion mastermix of Thermoscientific, see: Phusion mastermix product information
|
Component
|
Volume (in µL )
|
|
2xPhusion Mix
|
10
|
|
Forward primer
|
0.5
|
|
Reverse primer
|
0.5
|
|
gDNA
|
0.5
|
|
MilliQ
|
8.5
|
- Preheat lid (temp 99°C)
- Initial denaturation: 98 °C for 1 sec.
- For 34 cycles:
- Denaturation: 98 °C for 10 sec.
- Annealing: 45 °C for 20 sec.
- Extension: 72 °C for 1 min. (extension time depends on DNA fragment length: roughly 1 min per kbp)
- Final extension: 72 °C for 10 min.
- Storage: 4°C
- Gel purify the individual PCR fragments from 0.8% agarose gel. Determine the relative amounts of DNA for each fragment. This relation will be used to determine the volumes of the fragments in the phusion PCR mix.
- Phusion PCR
|
Component
|
Volume (in microL)
|
|
2xPhusion Mix
|
10
|
|
Fragment 1
|
...
|
|
Fragment 2
|
...
|
|
MilliQ
|
...
|
|
Total
|
20
|
- Preheat lid (temp 99°C)
- Initial denaturation: 98 °C for 1 sec.
- For 14 cycles:
- Denaturation: 98 °C for 10 sec.
- Annealing: 55 °C for 20 sec.
- Extension: 72 °C for 1 min. (extension time depends on DNA fragment length: roughly 1 min per kbp)
- Final extension: 72 °C for 10 min.
- Storage: 4°C
- Amplify the phusion PCR products using PCR as described in step 1 (using the forward primer of fragment 1 and the reverse primer of fragment 2)
- Check the results using gel electrophoresis with 0.8% agarose.
- Gel purify the PCR product.
All steps should be performed sterile near a flame
- Pipet 2-6 mL of culture medium (LB for E.coli, BG11 for Synechocystis) in a glass tube
- Add antibiotics if desired
- Scrape 1 colony from a plate containing the desired strain and stir the cells in the medium
- Incubate at 33°C at 200 rpm (E.coli) or at 37°C under high light conditions (Synechocystis)
Depending on the experimental design to be used, set the light intensity for every well by using the .csv file that corresponds to the experiment. These files are in concordance with the designs found here here.
- Intensity values are in uE.m-1.s-1
- Prepare spacer fragment by annealing oligos (phusion PCR)
- Add 5 μL of each oligo in a PCR tube
- Mix for 3 min at 95°C
- Cool to 4°C at a rate of 0.1°C/second
- Enzymatic digestion of the plasmid and incorporation of spacer fragment: Prepare mixture, use 1 μL of of annealed oligos mixture prepared in previous step (see Table for volumes)
- Ligate overnight at 4°C or for at least 2 hours ar 16°C
|
Reagent
|
Volume (μL)
|
|
10x buffer
|
0.5
|
|
T4 Ligase
|
0.5
|
|
pWD188
|
1
|
|
Annealed oligos
|
1
|
|
MilliQ H2O
|
2
|
|
Total Volume
|
5
|
All steps should be performed sterile near a flame
- add 0.5 µL of the plasmid DNA to E.coli competent cells from -80°C freezer (defrost on ice)
- Leave on ice for 30 min (for vector DNA to attach the to the cell wall)
- Heat shock for exactly 1 minute in a water bath at 42°C (time depends on cell volume)
- Add 900 µL of LB medium
- Incubate for 1 hour at 37°C while shaking
- Centrifuge the cells for 5 min at 5000 rpm. Dispose most supernatant
- Resuspend the cells in the remaining supernatant
- Spread the cells on an LB agar plate. This plate could contain the antibiotics for the selection of the cells which have taken up the plasmid.
- Incubate the plate for 1 day at 37°C
- Store the plates in the fridge (4°C)
All steps should be performed sterile near a flame
Make sure the following liquid cultures are made a day in advance:
Synechocystis
2 mL of E.coli with the following plasmids:
- Plasmid with elements for mobility (mob, for example pWD133) + desired plasmid
- Plasmid with elements for conjugation (prp4)
- Pipet 0.5-1 mL of the liquid culture into an eppendorf tube
- Centrifuge for 5 min at 5000 rpm. Dispose supernatant
- Wash 3x with 600 µL of LB (5 min at 5000 rpm) to remove any antibiotics from the medium
- Resuspend the cells in 100 - 200 µL of LB medium
- Transfer 100 µL of E.coli with the conjugation plasmid (prp4) to the eppendorf with E.coli containing the mob and desired plasmid.
- Clean the bench with ethanol
- Determine the OD of 800 µL Synechocystis culture by measuring the absorbance at 730 nm. Transfer 1 OD equivalent of culture in an eppendorf tube.
- Centrifuge 5 min at 5000 rpm. Remove supernatant
- Wash 3x with 600 mL of BG11 (5 min at 5000 rpm) when antibiotics are used in the culture. Otherwise, this step can be skipped.
- Resuspend in 100 µL BG11.
- Combine the E.coli prepared in step 5 with Synechocystis.
- Spread the samples on a BG11 plate without antibiotics and with a membrane for the conjugation to occur. Incubate for 1 day at 37°C degrees under high light conditions (specifics of incubator)
- Transfer the membrane to a plate with antibiotics (for which there is a resistance gene in the plasmid). Only cells with the plasmid are selected. Results can be obtained after 5-7 days.
1. Prepare template DNA by phusion PCR containing +- 800 bp upstream and downstream a gene. If a knockin is desired, the sequence of the gene of interest should be inserted between the up- and downstream gene region.
2. Design gRNA which will guide the Cpf1 protein to the chromosomal gene which needs to be modified.
3. Insert the gRNA in a plasmid containing CRISPR-Cpf1 elements and an origin of transfer (for example pWD188)
4. Transform this E.coli with the following combinations of plasmids
- Original plasmid (without gRNA) + mobility plasmid (positive control)
- Plasmid with gRNA + mobility plasmid (sample and negative control)
- Conjugation plasmid (PRP4)
5. Perform a conjugation of the three following combinations:
- Positive control + PRP4
- Negative Control + PRP4
- Sample + PRP4 + template DNA
6. After 5-7 days the following number of colonies should have grown:
- Positive control: many colonies
- Negative control: few/no colonies
- Sample: few/some colonies
The first thing you need when writing an algorithm is a programming language in which you can write your code. We are writing in Python, one of the more popular programming languages. Python is straightforward and relatively easy to use for new programmers. This language was also known to most members of the team.
We are using Python for the development of our program. Python is a widely used programming language which aims to write easy readable, clear code in an object oriented manner. Python enables us to design Forbidden FRUITS in a modular fashion, using object oriented programming.
To make our code better readable and usable for other users we are adhering to the PEP 8 guideline for coding.
GitLab is an open source software development platform which enables us to share our work among the members of the team and our supervisors. It has built-in functions like version control, issue tracking, code review and which enabled us to see where your teammates are working on.
