Team:Tongji China/Protocol


Tongji iGEM - Protocols
Tongji iGEM
TongJi iGEM
Protocols
This is how we did our experiments.
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Introduction

Our project aims to control Drosophila’ courtship behavior by overexpressing dopamine in their brain. In our plan, we will make 7 parts, 3 plasmids with a specific promoter.
First of all, we need to design the PCR primers with Primer Premier 5. Then we run pfu PCR to clone our target gene out of the Drosophila’ genome. After that, we do the digestion of vector pUAST-3xHA and do the ligation by In-fusion cloning, then do the transformation. The last step in molecular construction is plasmid extraction.
Then we come to the Drosophila’ part, which includes microinjection, screening, testing parts by rtPCR and behavioral experiments.
All the details are shown below.

Plasmid Construction

PrimeSTAR® PCR
Mixture:
Reaction Components PCR Reaction
Template 5-200 ng
Primer-Forward (for In-Fusion Cloning) 1 μL
Primer-Reverse (for In-Fusion Cloning) 1 μL
PrimeSTAR® Max Premix (2x) 25 μL
ddH2O Add to 50 μL
Total Volume 50 μL

PCR Procedure:
Method Time
98°C pre-denaturation 10s
98°C denaturation 5s   35 cycles
55-65°C anneal 5s or 15s
72°C extend 5s/kb
4°C save end


PCR clean-up
Kit: NucleoSpin® Gel and PCR Clean-up
1. Mix 1 volume of sample with 2 volumes of Buffer NTI;
2. Place a NucleoSpin® Gel and PCR Clean-up Column into a Collection Tube (2 mL) and load up to 700 μL sample, Centrifuge for 30 s at 11,000 x g. Discard flow-through and place the column back into the collection tube;
3. Add 700 μL Buffer NT3 to the NucleoSpin® Gel and PCR Clean-up Column. Centrifuge for 30 s at 11,000 x g. Discard flow-through and place the column back into the collection tube. Centrifuge for 1 min at 11,000 x g to remove Buffer NT3 completely. Make sure the spin column does not come in contact with the flow-through while removing it from the centrifuge and the collection tube;
4. Place the NucleoSpin® Gel and PCR Clean-up Column into a new 1.5 mL micro centrifuge tube (not provided). Add 15–30 μL Buffer NE and incubate at room temperature (18–25 °C) for 1 min. Centrifuge for 1 min at 11,000 x g.

Digestion
Mixture:
Reaction Components Digestion Reaction
Vector 5 μg
Enzyme (NEB) 5 μL
Enzyme buffer (10x) 5 μL
ddH2O Add to 50 μL
Total Volume 50 μL
Digest vector for 16 h at 37°C.

Gel extraction
1. Take a clean scalpel to excise the DNA fragment from an agarose gel. Remove all excess agarose. Determine the weight of the gel slice and transfer it to a clean tube. For each 100 mg of agarose gel <2% add 200 μL;
2. Incubate sample for 5–10 min at 50 °C. Vortex the sample briefly every 2–3 min until the gel slice is completely dissolved;
3. Place a NucleoSpin® Gel and PCR Clean-up Column into a Collection Tube (2 mL) and load up to 700 μL sample, Centrifuge for 30 s at 11,000 x g. Discard flow-through and place the column back into the collection tube;
4. Add 700 μL Buffer NT3 to the NucleoSpin® Gel and PCR Clean-up Column. Centrifuge for 30 s at 11,000 x g. Discard flow-through and place the column back into the collection tube. Centrifuge for 1 min at 11,000 x g to remove Buffer NT3 completely. Make sure the spin column does not come in contact with the flow-through while removing it from the centrifuge and the collection tube;
5. Place the NucleoSpin® Gel and PCR Clean-up Column into a new 1.5 mL micro centrifuge tube (not provided). Add 15–30 μL Buffer NE and incubate at room temperature (18–25 °C) for 1 min. Centrifuge for 1 min at 11,000 x g.

In-Fusion Cloning
Kit: abm® Ligation-Free Cloning System
Mixture:
Reaction Components Cloning Reaction
Purified DNA insert 10-200 ng
Linearized Vector 50-200 ng
5x Ligation-Free Cloning Master Mix 4 µL
Nuclease Free H2O Add to 20 µL
Total Volume 20 µL
Note: The molar ratio between the vector and insert should be 1:3. This can be calculated as follows:
The optimal amount of cloning vector = [0.02 x cloning vector base pair number] ng (0.03 pmol).
The optimal amount of insert = [0.06 x insert base pair number] ng (0.09 pmol).
Mix well and incubate on ice for 30 min.

Transformation
1. Get competent E.coli from -80℃ fridge;
2. Add 10μL plasmid liquid into competent E.coli, put it on ice for 30 min;
3. Give it a 42℃ heat shock for 90 sec.;
4. Put it into ice water for 3 min;
5. Add 200 µL LB into competence E.coli, 37℃, culture (220 rpm) for 45min;
6. Add 100 µL into a LB plate (Ampicillin). Inverted culture in the 37°C incubator for 14h-16h.

Colony PCR
Picked up single colonies in 1.5 mL EP tubes containing 200 μL LB (ampicillin), 37°C culture (220 rpm) for 3h.
Mixture:
Reaction Components PCR Reaction
Bacteria liquid 1 μL
2x Taq PCR Master Mix 5 μL
Primer-Forward 0.2 μL
Primer-Reverse 0.2 μL
ddH2O 3.6 μL
Total Volume 10 μL

PCR Procedure:
Method Time
95°C pre-denaturation 5m
95°C denaturation 30s   35 cycles
55-65°C anneal 30s
72°C extend 1m/kb
4°C save end


Plasmid Extraction
Kit: TIANprep Rapid Mini Plasmid Kit
1. Put the bacteria liquid in the EP tube, Centrifuge at 13,000 x rpm for 1 minute, discard the supernatant;
2. Add 150 μL P1 into the tube, vortex it;
3. Add 150 μL P2, mix the liquid upside down gently for 6-8 times until it appears clear purple;
4. Add 350 μL P5, mix the liquid upside down for 12-20 times until it appears yellow;
5. Stand for 5 minutes, then centrifuge at 13,000 x rpm for 5 minutes;
6. Transfer the supernatant into a clean DNA Mini Column CP3 with a 2mL collection tube (provided), centrifuge at 13,000 x rpm for 1 minute, discard flow-through and place the column back into the collection tube;
7. Add 300 μL PWT to the column, centrifuge at 13,000 x rpm for 2 min at room temperature to pass solution through column, discard flow-through and place the column back into the collection tube;
8. Centrifuge again at 13,000 x rpm for 1 min at room temperature;
9. Place column into a new clean 1.5mL tube. Add 50 μL 50°C ddH2O to the column and centrifuge at 13,000 x rpm for 2 minutes to elute DNA.

Behavioral Tests

Material
Canton.S, a kind of wild fruit fly from Canton, was used as control group. W1118 is a kind of fruit fly with a genetic mutation in the white gene. We transferred the plasmid to the flies and use them as treated group.
In the screening process after microinjection, the red eye gene W+ in the plasmid was used to determine whether the plasmid successfully inserted the chromosome. We picked the red eye fruit fly from the offspring. Then we interbred the red eye flies with the Cyo/Sp;Tb/Sb white eye flies to show which chromosome this gene was integrated into.
The relationship between genes and phenotypes is as follows: Cyo/curly wing, Sp/hairy, Tb/short body, Sb/short hair. The Cyo gene and Sp gene are located in the second pair chromosome of flies while the Tb gene and Sb gene are located in the third pair chromosome of flies. The homozygote of these genes is fatal. In the flies we use, these four genes are located on different chromosomes.
The hybridization is as follows:
NO DESCRIPTION
P means the parent (red eye fruit fly). P/P mean the second/third chromosome from the parent (red eye fruit fly). By looking at the color of P/Cyo;Tb/Sb and Cyo/Sp;P/Tb fruit fly's eyes, we can know which chromosome the gene was integrated into. And in these flies, the gene is steadily hereditary. Then we reserve fruit flies that have been transferred to the target gene for subsequent experiments.


Experiment 1: Use real-time PCR to determine whether the expression of TH is higher at 29°C

PROTOCOL

A) RNA extraction
Kit: Promega Eastep○R Super RNA extraction
1. Preparation of pyrolysis: Take 10 drosophila/head tissues. The tissue was quickly put into a 1.5ml EP tube without nucleic acid enzyme. Add the RNA lysis fluid, ice bath, and the tissue homogener were used to break the cell.
2. Add RNA diluent to the cleft and mix well with a pipette. Place for 3-5 minutes at room temperature.
3. Centrifuge the tubes for 5 minutes at maximum centrifugal speed at room temperature.
4. Pipette the upper cleaning liquid to the new 1.5ml tube without nucleic acid enzyme carefully.
5. Add anhydrous ethanol, whose volume is 0.5 times of the upper cleaning liquid, and blow 3-4 times with pipette to mix well.
6. Take out the corresponding number of centrifugal column/collection tube according to the number of samples (the centrifugal column has been placed on the collection tube).
7. Transfer the mixture to the centrifugal column.
8. 12,000-14,000 x g, centrifuge for 1 minute. Discard the filtrate and put the centrifugal column back into the collection tube.
9. Add 600µl RNA lotion (with ethanol) to the centrifugal column, 12,000-14,000 x g, centrifuge for 45 seconds, discard filtrate.
10. Digestion it with DNA enzyme I. To take a nucleic acid enzyme EP tube and add the following reagents: 10XDNA enzyme I buffer (5μl), DNA enzyme I (5μl) and Water without nucleic acid enzyme (40μl). Gently suck and mix well, don't oscillate.
11. Add 50µl DNA enzyme I incubation solution to the center of the centrifugal column, and leave it for 15 minutes at room temperature.
12. Add 600µl RNA lotion to the centrifugal column, 12,000-14,000 x g, centrifuge for 45 seconds, and discard filtrate.
13. Add 600µl RNA lotion to the centrifugal column, 12,000-14,000 x g, centrifuge for 45 seconds, and discard filtrate. The centrifugal column was resettled on the collection tube, with 12,000-14,000 x g centrifuge for 2 minutes.
14. Transfer the centrifugal column to the elution pipe, add 50µl water without nuclease in the middle of the membrane of column centrifugal, leave it for 2 minutes at room temperature, 12000-14000 x g, centrifuge for 1 minute and save RNA in - 70°C.

B) Reverse transcription

Wild Type Modified fruit fly
RNA 400ng 400ng
5×prime script 2µl 2µl
H2O Add to 10µl Add to 10µl
37°C for 15min; then 85°C for 5s
Add H2O to 40µl.

C) rtPCR

WT(internal reference) WT(TH) Modified fruit fly(internal reference) Modified fruit fly(TH)
DNA 4µl 4µl 4µl 4µl
SyBrⅡ 10µl 10µl 10µl 10µl
dH2O 4.4µl 4.4µl 4.4µl 4.4µl
Primer(tublin) 0.8µl×2 0 0.8µl×2 0
Primer(TH) 0 0.8µl×2 0 0.8µl×2


Segment1 95°C 5min
Segment2 (40 cycles) 95℃ 30s
55℃ 30s
72℃ 30s
Segment3 95℃ 1min
55℃ 30s
95℃ 30s



Experiment 2: Detect male-male courtship when raising the temperature [n=5]

PROTOCOL
(1) (2)
Group A One modified male fruit fly One wild-type male fruit fly
Group B One modified male fruit fly One wild-type male fruit fly

1. Group A was cultivated in 25℃ incubator and group B was cultivated in 29℃ incubator.
2. Both group A and B were placed in the box that can control circadian rhythm for 3 days before experiment.
3. Put one wild-type male fly in each of the four observation rooms.
4. Two groups of flies were transferred to the four separate observation room under light CO2 anesthesia and marked.
5. Use a digital camera to take the record (5 minutes) and calculate the courtship index.
The courtship index (CI) is the percentage of time that the fruit fly use for mating in five minutes.


Experiment 3: Test the modified Drosophila’ gender preferences [n=8]

PROTOCOL
1. Cultivate the modified male fruit fly in 29℃ incubator.
2. The male fruit flies were placed in the box that can control circadian rhythm for 3 days before experiment.
3. Put one decapitated wild-type male fly and one decapitated wild-type female fly in each of observation rooms.
4. Transfer one modified male fruit fly to each separate observation room under light CO2 anesthesia and marked.
5. Use a digital camera to take the record (10 minutes) and calculate the preference Index.
Preference Index (PI) was calculated according to the following equation: PI = Tm/(Tf +Tm), where Tf and Tm represent the time courter spent courting the female and the male in 10 min, respectively.


Experiment 4: Test whether the modified fruit flies increase their appeal for males [n=5]

PROTOCOL
Group A One wild-type male fruit fly
Group B One wild-type male fruit fly

1. Both group A and B were cultivated in 29℃ incubator.
2. Both group A and B were placed in the box that can control circadian rhythm for 3 days before experiment.
3. Put one decapitated modified male fly in observation room A and one decapitated wild-type male fly in observation room B.
4. Group A was transferred to the observation room A and group B was transferred to the observation room B under light CO2 anesthesia and marked.
5. Use a digital camera to take the record (5 minutes) and calculate the courtship index.


Experiment 5: Does the co-culture of wild-type and modified fruit flies influence their courtship with wild-type females? [n=5]

PROTOCOL
Group A One wild-type male fruit fly and one modified male fruit fly
Group B Two wild-type male fruit fly

1. Both group A and B were cultivated in 29℃ incubator.
2. Both group A and B were placed in the box that can control circadian rhythm for 3 days before experiment.
3. Put one virgin fly modified male fly in each of observation rooms.
4. Take out the modified male flies from the group A and one male fruit fly from group B. Then the rest of flies was transferred to the separate observation room under light CO2 anesthesia and marked.
5. Use a digital camera to take the record (5 minutes) and calculate the courtship index.


Experiment 6: Is the viability of modified fruit flies affected?

PROTOCOL
Group A Ten modified male fruit flies
Group B Ten wild-type male fruit flies

Group A and group B were cultured under the same conditions.
Then change only one factor in each experiment:
(1) lack food and water;
(2) high temperature(37°C);
Record the number of live fruit flies at regular intervals.
(3) the seismo-tube experiment
The machine vibrates the vertical tubes every 1 minute, leaving the fruit fly at the bottom of the culture tube. Observe the height of the flies’ crawling in 10 seconds.


Experiment 7: The reproductive curve of the modified fruit fly. [n=5]

PROTOCOL
1. Put one virgin fly and one modified male fruit fly in each culture tube.
2. All flies were cultivated in 29℃ incubator for 24h.
3. Take out the female flies from all groups and cultivate them separately.
4. The female flies were transferred to the new culture tube daily.
5. Wait a few days; record the number of larvae every day.


Experiment 8: Does the proportion of modified flies have an impact on mating rates of population? [n=5]

PROTOCOL
(1) Five modified male flies and five wild-type female flies
(2) Four modified male flies, one wild-type male fly and five wild-type female flies
(3) three modified male flies, two wild-type male flies and five wild-type female flies
(4) two modified male flies, three wild-type male flies and five wild-type female flies
(5) one modified male fly, four wild-type male flies and five wild-type female flies
(6) Five wild-type male flies and five wild-type female flies

All group were cultivated in 29℃ incubator for 24h.
Take out the female flies from all groups and cultivate them separately. Observe the fruit fly spawning to determine whether the flies mated and calculate mating rate.
Ignis Fly

Tongji_China iGEM 2017 Team
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