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From 2011.igem.org

Contents 1 Measurement 1.1 Solubilization of Antibiotics 1.2 Transformation 1.2.1 Sequence 1.2.2 Measurement of RPU 1.2.3 preliminary experiments 1.3 Assay of drosophila's phototaxis in different colors 1.3.1 1. Apparatus 1.3.1.1 Y maze 1.3.2 Pooter(吸虫管) 1.4 2. Drosophilas 1.4.1 3. Method

Measurement

Solubilization of Antibiotics

1. Mix the following (Final concentration is 50mg/mL).
   • Ampicillin:

     Ampicillin

     1.0g

     MilliQ

     20mL

   • Kanamycin:

     Kanamycin

     0.5g

     MilliQ

     10mL

2. Dispense 1.1mL of the solution into 1.5mL tubes.
3. Store in the -20℃ freezer.

Transformation

1. Unfreeze conpitent cells on ice.
2. Dry a plate by letting the plate upside down and partly open in incubator.
3. Add 1µL DNA solution and 20µL compitent cells to 1.5mL tube, let stand for 30min on ice. If few colony is observed, increase the amount of the compitent cells or DNA, but make the amount of DNA not to get over that of the compitent cells.
4. Heatshock for 60s at 42℃.
5. Let stand for 2min on ice.
6. Culture for 1h in preculture medium (LB or SOC medium), and plate by using spreader. Do not heat spreader too much because e.coli will dead for heat.

Sequence

1. Use Big Dye Terminator 3.1（ABI）
2. Mix the following

   5xBuffer

   2µL

   Primer (3.2µM)

   1µL

   Template Plasmid 

   200ng

   Big Dye Terminator 3.1

   0.5µL

   MilliQ

   up to 10µL

3. Let stand for 1min at 96℃.
4. 35 cycles for 5s at 98℃, for 5s 50℃, and for 2.5min at 68℃.
5. Add 25µL 100% ethanol and 1µL NAOAC

Measurement of RPU

1. Cultivate E.coli in M9 media(+ casamino acids) for about 15 hours.
2. Dispence 2.4ml to each tube.
3. Centrifuge these tubes (13,000 rpm , 4℃, 1min) and discarded the supernatant.
4. Add 1.2ml media(- casamino acids) and centrifuged at 4℃ twice.
5. Centrifuge these tubes again and discarde the supernatant and added 1.2ml media(-casamino acids) at 37 ℃.
6. Bring up E.coli at 0,5,10,15,20,25,30,60min and extracted RNA and synthesized cDNA.

preliminary experiments

1-1:plotting the standard line

Sample	glucose solution(various concentration from 0.20 mM to 1.60 mM)
Blank	mixed liquid (240μℓDNS reagent plus 1760㎕distilled water　)

1. added 240㎕ DNS reagent to 80㎕sample
2. heated it for 5 min in boiling water and then cooled it in water.
3. added it distilled water by 2ｍｌ
4. measured absorbance in 550nm

The standard curve is shown in fig 1.

1-2:evaluate the affection of medium

we examined the interruption based on the components of media(SOC, plas-grow and M9)

Sample	each medium
Blank	mixed liquid (240μℓDNS reagent plus 1760㎕distilled water　)

1. added 240㎕ DNS reagent to 80㎕sample
2. heated it for 5 min in boiling water and then cooled it in water.
3. added it distilled water by 2ｍｌ
4. measured absorbance in 550nm

The result is shown in fig 2.

1-3:measurement the effect of remainded E.coli

Sample	each medium
Blank	mixed liquid (240μℓDNS reagent plus 1760㎕distilled water　)

This assay was performed three times for each medium

1. poured the medium　cultured E.coli overnight 1.2 ㎕ into each five microcentritube.
2. centrifuged them for 5 min at 5,000 rpm
3. prepared new five microcentritube and move 800㎕ the supernatants into each of them.
4. measure the OD550 of one tube（use fresh medium as a blank in following assays）
5. one hour after, we measured OD 550 of other tube
6. take 80㎕ supernatant and move it into new tube and then heated it for 3 min in boiling water and then cooled it in water.
7. added 240㎕ DNS reagent and heated it for 3 min in boiling water and then cooled it in water.
8. two, three, five hours after, we did above operation, taking supernatant, measured OD500, heating and cooling, applying DNS reagent and heating and cooling again.
9. added all sample tube (containing 320㎕ solution) distilled water by 2ｍｌ and measure the absorbance of them in 550nm.

The results of measurement OD550 are shown in fig 3 and of measurement DNS assay are shown in fig 4.

Assay of drosophila's phototaxis in different colors

1. Apparatus

Y maze

Y maze was constructed by a Y joint, 弁, straws and LEDs.弁 was made of a blue tip of pipetman and it had the function to prevent drosophilas to go backward. Straws and 弁 were fitted with the two terminals of Y joint by vinyl tape as figure.LEDs were fitted with the other terminals of the straws by 凹-form parts made of polyethylene board. The angles of Y-form was 120°.

This Y maze is put in the dark-room environment. (作成者：草場、未添削)

Here is the list of the LEDs which we used in the experiment.

The list of our LEDs

Color	Manufacturer	No.	Name	Wavelength
Ultraviolet	OptoSupply	OSSV5111A	5mm Super Violet LED	Peak wavelength = 400 nm
Blue	OptoSupply	OSUB5161P	5mm Deluxe Blue LED	Dominant wavelength = 465~475 nm
Green	SHARP	GL3GC402B0SC	High-luminosity(InGaN) LED Lamp	Dominant wavelength = 525 nm
Red	OptoSupply	OSHR5161P	5mm Deluxe Red LED	Dominant wavelength = 620~630 nm
Infrared	OptoSupply	OSIR5113A	5mm Infrared Emitting Diode	Peak wavelength = 940 nm

Pooter(吸虫管)

A pooter is very useful tool to move drosophilas. We also used it to move them from rearing bottles to the Y maze.

Our pooter consisted of two parts: tube part and tip part. The tube part was made of vinyl tube and two cutted tips of pipetman. The cutted tips were fitted with the terminals of tube by vinyl tape as mouthpiece and objective end. The tip part was made of two cutted tips of pipetman and a piece of tissue paper. Not to inhale drosophilas into the mouth, the piece of tissue paper was slipped in between the cutted tips.

When we move drosophilasfrom rearing bottles to the Y maze, we fitted the tip part with the tube part and draw some drosophilas into the tip part by mouth. Then closing the orifice of tip part by finger, we removed the tip part from the tube part and put it into the Y maze.(作成者：草場、未添削)

2. Drosophilas

We used the Oregon-RS of Drosophila melanogaster. They were derived from Dr. Fuse's laboratory on 4 August 2011. They were maintained in an incubator at 22℃ on standard cornmeal medium in the ordinary glass bottle (____cm high, ____cm in diameter) with a 12h light phase (12:12 light:dark cycle). Flies were aged 2-4 days after emergence.

3. Method

Firstly, we gathered 5 flies with a pooter. After that, we removed the tip of the pooter and attached the tip to the bottom end of the Y maze. Then we left the flies intact in the dark for three minutes and counted the number of flies in each tube. In this experiment, we switched both the left and right LEDs on for 2 times alternatively. We ran the experiment 4 times using 2 sets of 5 male flies and 2 sets of 5 female flies. We summed up the number of flies that entered the lighting LED end and opposite end and ran the t test.

ハエ5匹を吸虫管で取る。吸虫管チップ部を取り外し、Y mazeの下部に差し込み、取り付ける。暗黒下で3分間放置し、Y　mazeのどちらの管に何匹入ったかをカウントした。実験は左のライトを点灯した場合を2回、右のライトを点灯した場合を2回測定した。オス5匹1セットを2組、メス5匹1セットを2組行い、ライト点灯側と反対側に入ったハエの数のそれぞれ合計をとり、t検定を行った。(by　草場)