Team:SHSID/Experiments
Experiments
LB Media Preparation
Materials:
1. Yeast extract 5g/L (0.5%)
2. Tryptone 10g/L (1%)
3. NaCl 10g/L (1%)
4. Add agar 15g/L (1.5%)
5. Distilled water 600mL
6. 4 150mL Beakers
7. Graduated Cylinder
2. Tryptone 10g/L (1%)
3. NaCl 10g/L (1%)
4. Add agar 15g/L (1.5%)
5. Distilled water 600mL
6. 4 150mL Beakers
7. Graduated Cylinder
Methods:
1. Measure 600 mL of distilled water & add into a big beaker
2. Measure 6g of tryptone, add to beaker
3. Measure 6g of NaCl, add to beaker
4. Measure 3g of yeast extract, add to beaker
5. Stir and dissolve completely using a stirring rod
6. Prepare 4 150 mL beakers
7. Measure and add 2.25 g of agar (per flask) to two of the four flasks
8. Distribute solution to the four flasks, 150 mL each.
9. Seal using brown paper & rubber band as lid
10. Put on sterilization tape
11. Autoclave for with the setting of 121oC for 30 minutes (Total duration 2 hours)
2. Measure 6g of tryptone, add to beaker
3. Measure 6g of NaCl, add to beaker
4. Measure 3g of yeast extract, add to beaker
5. Stir and dissolve completely using a stirring rod
6. Prepare 4 150 mL beakers
7. Measure and add 2.25 g of agar (per flask) to two of the four flasks
8. Distribute solution to the four flasks, 150 mL each.
9. Seal using brown paper & rubber band as lid
10. Put on sterilization tape
11. Autoclave for with the setting of 121oC for 30 minutes (Total duration 2 hours)
YPD Media Preparation
Materials:
1. Yeast extract 20g/L
2. Tryptone 40g/L
3. Glucose (independent liquid, 40g/L medium concentration)
4. If making solid, add agar 15g/L
5. Distilled water 500mL
6. 2 100mL beakers, 1 300mL beaker
2. Tryptone 40g/L
3. Glucose (independent liquid, 40g/L medium concentration)
4. If making solid, add agar 15g/L
5. Distilled water 500mL
6. 2 100mL beakers, 1 300mL beaker
Methods:
1. Measure 500ml water, add to beaker
2. Measure 20g tryptone, add to beaker
3. Measure 10g yeast extract, add to beaker
4. Mix all substances to dissolve, use magnetic stirring if needed
5. Distribute solution to 300ml, 100ml, and 100ml triangular flask
6. Add 1.5g agar to 100ml to make solid
7. Use brown paper to seal the triangular flask, wrap with rubber band
8. Put on sterilization tape
9. Autoclave at 121oC for 30min, total time 2hrs
2. Measure 20g tryptone, add to beaker
3. Measure 10g yeast extract, add to beaker
4. Mix all substances to dissolve, use magnetic stirring if needed
5. Distribute solution to 300ml, 100ml, and 100ml triangular flask
6. Add 1.5g agar to 100ml to make solid
7. Use brown paper to seal the triangular flask, wrap with rubber band
8. Put on sterilization tape
9. Autoclave at 121oC for 30min, total time 2hrs
Prepare competent cell solution
Materials:
*** glycerol 20% *** total 80ml solution
1. glycerol 16ml
2. Distilled water 64ml
2. Distilled water 64ml
Methods:
1. Measure 64ml water, add to cylinder
2. Add oil 16ml
3. Add water until solution reaches 80ml
4. Mix thoroughly, transfer to bottle with cap, seal with sterilization tape
2. Add oil 16ml
3. Add water until solution reaches 80ml
4. Mix thoroughly, transfer to bottle with cap, seal with sterilization tape
PEG 3350 Solution
Materials:
1. PEG 25g
2. Distilled water 15ml
2. Distilled water 15ml
Methods:
1. Measure 25g PEG, add to cylinder
2. Add distilled water 15ml
3. Stir then heat until dissolve (becomes transparent solution)
4. Transfer to bottle and seal
5. Put on sterilization tape
6. Autoclave at 121oC for 30min, total time 2hrs
2. Add distilled water 15ml
3. Stir then heat until dissolve (becomes transparent solution)
4. Transfer to bottle and seal
5. Put on sterilization tape
6. Autoclave at 121oC for 30min, total time 2hrs
Lithium Acetate Solution
Materials:
1. 5.1g of lithium acetate
Methods:
1. Measure 5.1g of lithium acetate
2. Measure 50mL of distilled water
3. Add lithium acetate in water, stir and dissolve.
4. Close apparatus lid, making sure that the lid is not completely sealed
5. Autoclave for 30 minutes at 121oC (total duration 2 hours)
2. Measure 50mL of distilled water
3. Add lithium acetate in water, stir and dissolve.
4. Close apparatus lid, making sure that the lid is not completely sealed
5. Autoclave for 30 minutes at 121oC (total duration 2 hours)
Culture Medium
1. YPD → yeast
ingredients: yeast extraction(20g/L); tryptone (40g/L); glucose (200g/L originally and 40g/L，added 400µg)
2. LB → e. coli
a. ingredients: yeast extraction(5g/L); tryptone (10g/L); sodium chloride (10g/L)
Fill in Petri dish
combine with 2 antibiotics
LB uses amp（100mg/mL, to dilute to 100 µg /mL we pour 150 µL using pipet）
YPD uses HYG (200/mL, to dilute to 400 µg /mL we pour 200 µL using pipet）
Inoculum and culture
1. speb (E coli)
2. Yeast plasmid (pYES2) (4mL of broth, 母液100mg/mL target 100 µg /mL, pipet 4 µL)
3. by4741 (10 µL yeast)
shaker bed incubator 30⁰C 200rpm overnight
2. Yeast plasmid (pYES2) (4mL of broth, 母液100mg/mL target 100 µg /mL, pipet 4 µL)
3. by4741 (10 µL yeast)
shaker bed incubator 30⁰C 200rpm overnight
PCR
1. DNA replication
2. In vivo DNA replication process:
2. In vivo DNA replication process:
central dogma: DNA → RNA → protein
semi-conservative replication
one parent strand, one new strand
1. helicase: unzips two strands of DNA
2. primase: synthesize RNA primer for DNA polymerase III to attach
3. DNA polymerase III: add complementary nucleotides at the 3' end
4. DNA polymerase I & DNA ligase: replace primer & combine okazaki fragments
semi-conservative replication
one parent strand, one new strand
1. helicase: unzips two strands of DNA
2. primase: synthesize RNA primer for DNA polymerase III to attach
3. DNA polymerase III: add complementary nucleotides at the 3' end
4. DNA polymerase I & DNA ligase: replace primer & combine okazaki fragments
3. In vitro DNA replication process:
Denaturing: heat to 95-98⁰C, break hydrogen bonds and separate DNA into two strands
Annealing: DNA sample is cooled to 54 ⁰C (generally 50-60), depending on the length of primers, allowing
forward and reverse primers attach to opposite ends of the target DNA sequence
ATCG (Adenine, Thymine, Cytosine, Guanine)
number of hydrogen bonds (2, 3 → different temperature)
Extending: A heat-tolerant DNA polymerase (Taq) added nucleotides at 72 ⁰C
5' end to 3' end
2^30 DNA by 30 rounds of replication
Annealing: DNA sample is cooled to 54 ⁰C (generally 50-60), depending on the length of primers, allowing
forward and reverse primers attach to opposite ends of the target DNA sequence
ATCG (Adenine, Thymine, Cytosine, Guanine)
number of hydrogen bonds (2, 3 → different temperature)
Extending: A heat-tolerant DNA polymerase (Taq) added nucleotides at 72 ⁰C
5' end to 3' end
2^30 DNA by 30 rounds of replication
4. system
2x PCR mix 10 µL includes:
buffer
dNTP, ATP, TIP, CTP, GTP
Taq, DNA polymerase
primer - F 0.2 µL
primer - R 0.2 µL
template 1 µL
ddH2O 8.6 µL
buffer
dNTP, ATP, TIP, CTP, GTP
Taq, DNA polymerase
primer - F 0.2 µL
primer - R 0.2 µL
template 1 µL
ddH2O 8.6 µL
5. program
Plasmid Extraction
Agarose gel el electrophoresis
Materials:
1. agarose N
2. TAE buffer
3. Loading buffer
4. nucleic acid dye
5. DNA samples after PCR
2. TAE buffer
3. Loading buffer
4. nucleic acid dye
5. DNA samples after PCR
Methods:
1. Dissolve 1g agarose in 100 mL TAE buffer (1% w/v), microwave for about 2 minutes until mixture turns
transparent
2. Put small comb and medium-sized comb for identifying DNA and large comb for recycling DNA
3. Pour the agarose solution in slot, completely covering the large comb and covering half of the small comb
4. Wait for 30 minutes to cool and solidify
5. During waiting: prepare DNA sample
a. Loading buffer + 1 µL nucleic acid dye to visualize DNA under UV and sink to the bottom of gel under TAE buffer solution
b. 5 µL DNA sample + 1 µL buffer solution
6. Pour more TAE buffer in electrophoresis device so that the liquid covers the whole sample
7. Put the sample with appropriate order in each hole (First two are markers)
8. Set electrophoresis at 185V for 15 minutes
9. Use UV to visualize the DNA strand
2. Put small comb and medium-sized comb for identifying DNA and large comb for recycling DNA
3. Pour the agarose solution in slot, completely covering the large comb and covering half of the small comb
4. Wait for 30 minutes to cool and solidify
5. During waiting: prepare DNA sample
a. Loading buffer + 1 µL nucleic acid dye to visualize DNA under UV and sink to the bottom of gel under TAE buffer solution
b. 5 µL DNA sample + 1 µL buffer solution
6. Pour more TAE buffer in electrophoresis device so that the liquid covers the whole sample
7. Put the sample with appropriate order in each hole (First two are markers)
8. Set electrophoresis at 185V for 15 minutes
9. Use UV to visualize the DNA strand
Nanodrop
1. used nanodrop to measure the concentration of yeast inoculation in yeast solution
a. follow nanodrop steps as described in 6/28
2. undiluted yeast solution should have an Optical Density (OD) >10
a. OD estimated how much DNA you have in solution by measuring the absorbance at a certain wavelength
i. the most common wavelength used is 600 nm, which is one we used in our experiment
b. we first diluted the solution from 0 and found that our yeast solution's OD was 2
a. follow nanodrop steps as described in 6/28
2. undiluted yeast solution should have an Optical Density (OD) >10
a. OD estimated how much DNA you have in solution by measuring the absorbance at a certain wavelength
i. the most common wavelength used is 600 nm, which is one we used in our experiment
b. we first diluted the solution from 0 and found that our yeast solution's OD was 2
3. then we add glucose to the yeast solution for dilution
Prepare Yeast Competent Cell
1. culture yeast cells in 3mL test tubes
2. measure OD
a. pipette 100µL of yeast culture into 4mL of YPAD in culture flask
3. culture yeast strains for 3.5 hours, measure OD again (new OD is 0.3)
4. centrifugation
a. 4500 rpm for 5 minutes
5. use ddH2O to wash and re-suspend the cells
a. washing process: add ddH2O → mix together → centrifuge → pour away the fluid section
6. add frozen competent cell solution (FCCS) (1% of total volume)
a. ingredients
2. measure OD
a. pipette 100µL of yeast culture into 4mL of YPAD in culture flask
3. culture yeast strains for 3.5 hours, measure OD again (new OD is 0.3)
4. centrifugation
a. 4500 rpm for 5 minutes
5. use ddH2O to wash and re-suspend the cells
a. washing process: add ddH2O → mix together → centrifuge → pour away the fluid section
6. add frozen competent cell solution (FCCS) (1% of total volume)
a. ingredients
b. purpose：to preserve the yeast in the fridge
7. separate yeast culture to 100 µL tubes at -20 ℃
Overlap PCR
procedure as follows
Gel Recycle
Measurements:
Methods:
1. measure 2g agarose powder
2. measure 200ml TAE
3. add two components in a triangular flask
4. heat the flask in a microwave for 2 min at medium heat, continue until substance all dissolved
5. prepare gel models with large sample holes
6. add the agarose TAE solution to the gel models
7. set aside to cool
2. measure 200ml TAE
3. add two components in a triangular flask
4. heat the flask in a microwave for 2 min at medium heat, continue until substance all dissolved
5. prepare gel models with large sample holes
6. add the agarose TAE solution to the gel models
7. set aside to cool
Gel Extraction
1. Add 8µL loading buffer into three 4-7 tubes (50µL each)
2. gel electrophoresis: 185V 22min
a) add 8.3µL of loading buffer (10x) to PCR sample (total 60 µL)
b) 15k marker
3. collect DNA and add 500 µL of NTI buffer
i) 4-7 first tube: 0.06g
ii) 4-7 second tube: 0.23g
iii) 4-7 third tube: 0.20g
c) Heat it at 50℃ until all the contents melt
4. centrifuge for 30sec at 12000rpm
5. Pour the liquid in microcentrifuge tube back to the column and centrifuge for 30sec at 12000rpm
6. add 700 µL of NT3 to wash silica membrane, then centrifuge for 30sec at 12000rpm (twice)
7. Dry the membrane by centrifuging for 1min at 12000rpm
8. add 50℃ of ddH2O 20µL and 30sec 12000rpm
9. Use nanodrop to check concentration
a. 4-7 first tube: 23.8
b. 4-7 second tube: 22.4
c. 4-7 third tube: 26.4
We decided to use 4-7 first and second tube.
2. gel electrophoresis: 185V 22min
a) add 8.3µL of loading buffer (10x) to PCR sample (total 60 µL)
b) 15k marker
3. collect DNA and add 500 µL of NTI buffer
i) 4-7 first tube: 0.06g
ii) 4-7 second tube: 0.23g
iii) 4-7 third tube: 0.20g
c) Heat it at 50℃ until all the contents melt
4. centrifuge for 30sec at 12000rpm
5. Pour the liquid in microcentrifuge tube back to the column and centrifuge for 30sec at 12000rpm
6. add 700 µL of NT3 to wash silica membrane, then centrifuge for 30sec at 12000rpm (twice)
7. Dry the membrane by centrifuging for 1min at 12000rpm
8. add 50℃ of ddH2O 20µL and 30sec 12000rpm
9. Use nanodrop to check concentration
a. 4-7 first tube: 23.8
b. 4-7 second tube: 22.4
c. 4-7 third tube: 26.4
We decided to use 4-7 first and second tube.
Reflection
Due to the lack of results (unsuccessful E. Coli culture) from previous lab, we made 2 changes
1. double the amount of DNA fragment
a. decrease water volume since the total volume must maintain 20µL
2. decrease number of fragments for gel electrophoresis
a. previous lab: 3 fragments
b. current lab goal: 2 fragments
a. decrease water volume since the total volume must maintain 20µL
2. decrease number of fragments for gel electrophoresis
a. previous lab: 3 fragments
b. current lab goal: 2 fragments
Experiment:
double the amount DNA fragments
double the amount DNA fragments
1. remake DNA solution (specifics as follow)
2. add mixture(5µL) into E. Coli template (dH5a)
a. ratio 1:10
3. heat shock in dry bath
a. 40℃ for 90 seconds
b. principle: cell membrane becomes semi-permeable under sudden exposure to higher temperature allowing the DNA fragments to enter in E.coli template
4. add LB culture (600µL)
a. principle: makes e. coli competent stronger
5. put mixture in rotary shaker for 60 minutes at 2200rpm
6. Remove 700µL of E.coli culturing solution
7. Vortex the remaining solution with residue
8. Open a prepared Petri dish with LB culture
9. Evenly transfer the remaining solution to the Petri dish with a pipette
10. Discard the initial pipette head and change a new one, bend the new head
11. Gently spread the culturing solution with the bent Petri dish head until the surface is even and dry
12. Close the Petri dish lid and seal with laboratory film, transfer Petri dish to incubator
a. ratio 1:10
3. heat shock in dry bath
a. 40℃ for 90 seconds
b. principle: cell membrane becomes semi-permeable under sudden exposure to higher temperature allowing the DNA fragments to enter in E.coli template
4. add LB culture (600µL)
a. principle: makes e. coli competent stronger
5. put mixture in rotary shaker for 60 minutes at 2200rpm
6. Remove 700µL of E.coli culturing solution
7. Vortex the remaining solution with residue
8. Open a prepared Petri dish with LB culture
9. Evenly transfer the remaining solution to the Petri dish with a pipette
10. Discard the initial pipette head and change a new one, bend the new head
11. Gently spread the culturing solution with the bent Petri dish head until the surface is even and dry
12. Close the Petri dish lid and seal with laboratory film, transfer Petri dish to incubator
3 combinations of DNA segments for constructing plasmid
goal: reduce the number of fragments by homologous recombination to improve plasmid construction
elongation time is based on length
successful combination allows total fragment number to reduce from 3 to 2, improve the probability of plasmid constructions' success
combinations
0123+45, length:5k
4567+678, length:3.7k---success
45+678, length: 3.7k
materials are as following
elongation time is based on length
successful combination allows total fragment number to reduce from 3 to 2, improve the probability of plasmid constructions' success
combinations
0123+45, length:5k
4567+678, length:3.7k---success
45+678, length: 3.7k
materials are as following
E.coli Culture
Methods:
1. Remove 700microliter of E.coli culturing solution
2. Vortex the remaining solution with residue
3. Open a prepared Petri dish with LB culture
4. Evenly transfer the remaining solution to the Petri dish with a pipette
5. Discard the pipette head and change a new one, bend the new head
6. Gently spread the culturing solution with the bent Petri dish head until the surface is even and dry
7. Close the Petri dish lid and seal with laboratory film, transfer Petri dish to incubator
8. Leave it overnight and check results in the morning
2. Vortex the remaining solution with residue
3. Open a prepared Petri dish with LB culture
4. Evenly transfer the remaining solution to the Petri dish with a pipette
5. Discard the pipette head and change a new one, bend the new head
6. Gently spread the culturing solution with the bent Petri dish head until the surface is even and dry
7. Close the Petri dish lid and seal with laboratory film, transfer Petri dish to incubator
8. Leave it overnight and check results in the morning
Putrescine Yield Measurement
Methods:
1. At least 3 repeated small-scale metabolite production tests were carried out in YNB-SC medium (containing
metabolic raw materials)
2. The yeast colonies were inoculated into 300 μL of culture solution and incubated on a shaker at 30°C, 460 rpm and 80% relative humidity for 48 hours.
3. The culture was pelleted by centrifugation at 3500 × g for 5 minutes at 12°C, and 100–200 μL of the supernatant (containing putrescine) was removed for analysis.
4. The production of metabolites was analyzed using Agilent 1260 Infinity binary HPLC and LC-MS/MS.
2. The yeast colonies were inoculated into 300 μL of culture solution and incubated on a shaker at 30°C, 460 rpm and 80% relative humidity for 48 hours.
3. The culture was pelleted by centrifugation at 3500 × g for 5 minutes at 12°C, and 100–200 μL of the supernatant (containing putrescine) was removed for analysis.
4. The production of metabolites was analyzed using Agilent 1260 Infinity binary HPLC and LC-MS/MS.