Thanks:

Sponsors:

Beijing Normal University (BNU), is a public research university located in Beijing with strong emphasis on basic disciplines of humanities and sciences. It is one of the oldest and most prestigious universities in China. Our school supported us to our iGEM Journey and provided us adequate resources to complete the competition. Students of different colleges in our school took part in our team, and all these colleges supported us very much.

5.1 Cloning

PCR

• Reaction System:

  H₂O 38 \(\mu\)L

  10x Taq buffer 5 \(\mu\)L

  2.5mM dNTP 4 \(\mu\)L

  Primer 1 1 \(\mu\)L

  Primer 2 1 \(\mu\)L

  Template 0.5 \(\mu\)L

  Taq 0.5 \(\mu\)L

• Process:

  \(\begin{equation}\left. \begin{array}{lcl} {94°C\ 10min} \\ {94°C\ 30s} \\{58°C\ 30s} \end{array} \right\} Cycle\ 30\end {equation}\)

  94°C 10min

  94°C 30s

  58°C 30s

  72°C 2min30s

  72°C 10min

  10°C ---




Electrophoresis---Gel Purification

• Material:

  Agarose gel: 1% agarose dissolved in 1 x TAE + gelstain

• Protocol:

  We used gelstain to stain the DNA and imaged it in a Transilluminator.

  We used the gel extraction kit to get the objective fragment.




Digestion

50\(\mu\)L reaction system
Reagent	10x H buffer	EcoR I	Pat I	Plasmid	H2O
Dosage	5 \(\mu\)L	1.5 \(\mu\)L	1.5 \(\mu\)L	15 \(\mu\)L	27 \(\mu\)l

10\(\mu\)L reaction system
Reagent	10x H buffer	EcoR I	Pat I	Plasmid	H2O
Dosage	1 \(\mu\)L	0.3 \(\mu\)L	0.3 \(\mu\)L	3 \(\mu\)L	5.4 \(\mu\)L




Ligation

Ligation reaction system
Reagent	Cph8	PSB1C3	T4 buffer	T4 ligase
Dosage	14 \(\mu\)L	3 \(\mu\)L	2 \(\mu\)L	1 \(\mu\)L

5.2 Transformation

• Material:

LB liquid medium
Reagent	Tryptone	Yeast extract powder	NaCl
Dosage	10 g/L	5 g/L	10 \(\mu\)L




• Protocol:

  preparation of the competent cells

  20\(\mu\)L ligation product + 50\(\mu\)L cells

  Heatshock of E.coli BW25113(42°C,90s)

  Put on ice(2min)

  Add 800\(\mu\)L LB media and incubate for 1.5h(37°C, 150rpm)

  Centrifuge at 4000rpm for 1min and remove 900\(\mu\)L supernatant

  Resuspend the pellets using the left supernatant

  Spread plates(with ampicillin)

  Incubate for 12~16h(37°C)

5.3 Detetion

SDS-PAGE

• Materials:

  Gel	Tris-HCl	Acr/Bis 30%	SDS 10%	ddH2O	TEMED	AP 10%
  Stacking Gel(4%)	pH=6.8 500\(\mu\)L	500 \(\mu\)L	25 \(\mu\)L	1350 \(\mu\)L	2.5 \(\mu\)L	12.5 \(\mu\)L
  Running Gel(12%)	pH=8.8 1250 \(\mu\)L	2000 \(\mu\)L	50 \(\mu\)L	1675 \(\mu\)L	2.5 \(\mu\)L	25 \(\mu\)L
  Running Gel(18%)	pH=8.8 1250 \(\mu\)L	3000 \(\mu\)L	50 \(\mu\)L	675 \(\mu\)L	2.5 \(\mu\)L	25 \(\mu\)L

  Running Buffer
  Reagent	Tris-HCl	Glycine	(w/v) SDS
  Dosage	25 mmol/L	0.192 mol/L	0.1%

• Protocol:

  The SDS polyacrylamide gels are prepared in the so-called PerfectBlue™ Twin Double Gel System.

  After ensuring that the equipment is waterproof, the 12 % (or 18%) running gel is mixed and filled into the chamber. Pipetting about 1 ml of H₂O on top of the running gel to seal the gel.

  After polymerization, the remaining H₂O is removed and the 12 % stacking gel is filled on top. Insert a comb to create sample pockets.

  After the stacking gel also polymerized, 1 x running buffer is used to run the Double Gel System via the SDS gel.

  After loading the generated pockets with the samples, the stacking gel is run at 100 V and then running gel at 120 V.

Detection of Baiting Nematodes

• Materials:

  Pure chemical compound: linalool and limonene.

  Solvent: DMSO

  Dissolve the compounds with DMSO to set a series of concentration gradient of attractant.

• Protocol:

  Synchronize the nematodes.

  Divide the NGM solid medium (d = 6 cm) equally into two parts (drawing on the surface of the culture dishes).

  Put 50 \(\mu\)L compound of different concentration and DMSO as contrast respectively on the two parts. Make 3 repeats of each concentration.

  Flush nematode from the plate with M9 and inoculate 20 \(\mu\)L (about 30 nematodes) into the center of the medium.

  Incubate at 20°C for 1hr, and then place them at 4°C refrigerator for 1hr.

  Count the nematodes of each part under the stereoscope.

  Compare the results of different compound concentration and make a conclusion.

Detection of Killing Nematodes

Inoculate 5mL LB liquid mediums added Chl with experimental group E.coli BW25113 (transformed with rMpl or bace16 genes) and blank control group separately. Incubate at 37°C, 190 r.m.p for 3 hr.

Mix up 800\(\mu\)L control group and 200ul experimental liquid medium Inoculate 1mL mixture mentioned above to NGM plate. Incubate upside down at 37°C overnight.

Flush nematodes from the plate with M9, centrifugate (1500 r.p.m, 3min), abandon the supernatant and resuspend the precipitation with 3mL M9.

Add 500\(\mu\)L resuspended solution to the NGM plate inoculated with mixed E.coli as mentioned above as experimental group and NGM plate with blank E.coli as control group. 37°C incubate overnight.

Observe and compare the activation and size of nematodes of each group and draw a conclusion.

5.4 Nematode

• Materials:

• NGM medium:

Minimum Medium
Reagent	NaCl	Bacto peptone（BD 4059002)	Agar	ddH2O
Dosage	3 g	2.5 g/L	17 \(\mu\)L	975 mL

Prepare 1mol/L CaCl₂, 1mol/L MgSO₄ and phosphoric acid buffer meanwhile.

Phosphoric Acid Buffer
Reagent	KH2PO4	KHPO4	ddH2O
Dosage	108.3 g	35.6 g/L	1 L

Autoclave the above mentioned reagents with 120°C for 30 min and then water bath them to 65°C.

Dissolve the cholesterol with ethanol to 5 mg/mL.

Add 1ml cholesterol, CaCl₂, MgSO₄ and 25 mL phosphoric acid buffer into the minimum medium in order (all at 65°C, shake up).

Make plate with the NGM medium, storing at 4°C.




• OP50 E.coli deficit type (food of nematodes):

Streak inoculate OP50 on LB solid medium, Incubate at 37°C for 6 hr or overnight. Inoculate LB liquid medium with OP50 single colonies, and incubate (37°C, 220 r.p.m) overnight.

Cultivate

• The incubation condition of caenorhabditis elegans N2 wild type:

  Generally incubated at 20°C. Grow slowly at 16°C and grow fast at 25°C while egg laying amount declines.




• Protocol:

Inoculate NGM medium with 150~200 \(\mu\)L OP50, incubate at 37°C for 12hr. Cut down a square of NGM(about 1cm x 1cm) with nematodes.

Put the square on the NGM medium with OP50 and let the surface with nematodes adown to contact the medium.

Seal and incubate upside down at 20°C.

Watch the growth condition of nematodes under the stereoscope everyday and re-inoculate every 4 to 5 days to avoid the nematodes growing too densely.

For inoculating abundant nematodes rapidly, or changing plate to provide more food and better condition for them, we can flush them with M9 and centrifuge them with 1500 r.m.p for 1 min, and incubate the precipitate on a new plate.




Synchronization

• Materials:

M9 NS
Reagent	Na2HPO4	KH2PO4	NaCl	1M MgSO4	ddH2O
Dosage	6 g	3 g	5 g	1 mL	1 L

Bleach Buffer
Reagent	NaOCl	5M NaOH	ddH2O
Dosage	50 \(\mu\)L	100 \(\mu\)L	850 \(\mu\)L




• Protocols:

We have tried several methods and two of them succeed as mentioned below, we recommend the first one by comparison.

1. Flush and incubate

Flush the nematodes with M9 NS

Centrifugation (3000 r.m.p, 1min)

Abandon the supernatant, add 1 mL bleach buffer to the precipitation

Centrifugation (3000 r.m.p, 1min), abandon the supernatant

Transfer the precipitation (nematode eggs) to a NGM plate with OP50, then the eggs will incubate synchronously.

2. Directly pick

Transfer 30 nematodes that are under egg laying period to a new NGM plate with OP50. Generally, the egg laying nematodes can lay about 8 eggs per hour. Remove all nematodes after 2hr.

Incubate the plate at 20°C for 3d and therefor can get about 300 to 400 nematodes.

Note: all of the procedure outlined above must be conducted under sterile condition.




Isolate nematodes from soil

• modified Baermann funnel method:

1. Place a glass funnel (d = 10~15 cm) on the funnel stand linked with about 10cm rubber tube which is furnished with a flatjaw pinchcock.

2. Cut the soil sample into matchstick shape. Package 10 g(wet weight) with gauze and put it into the funnel, and then add water to immersion the sample.

3. After 4~24 hr, nematodes leave the plant tissue and swim in the water and ultimately precipitate at the bottom due to their gravity and hydrotaxis.

4. Open the flatjaw pinchcock, adopt about 5~15 mL liquid sample from the bottom which obtains most active nematodes of the soil sample. Check under the stereoscope and count nematodes, or centrifugate(1500 r) first and check the precipitation if the nematodes are too few.