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(Click here to the original story at the NSF website)

High-school biology teachers are delving deeper into the plant world with the help of plant biologists at the University of Missouri. Through professional development workshops, the teachers learn concepts in plant biology from research scientists and receive curricular materials aligned with state and national science teaching standards.

Caption: Teachers extract DNA from plants during a workshop.

Credit: Laurent Brechenmacher, University of Missouri

This program is unique in that it incorporates aspects of basic scientific research into an engaging plant biology program for teachers, and emphasizes an investigative approach for classroom learning. In addition, the program has the teacher participants return to the workshops so they can share their experiences and gain additional insight into plant biology.

Caption: Teachers gain insights into plant anatomy and physiology.

Credit: Deanna Lankford, University of Missouri

 

Teachers learn how to extract DNA from plant materials, examine nodule formation in soybeans roots inoculated with the bacterium Bradyrhizobium japonicum, and create biofuels from plant oil. The teachers receive background information and student-ready investigations for each of the concepts emphasized within the program. They also receive soybean seeds, planting materials and a light set to support implementation of the investigations when they return to their classrooms.

Caption: Teacher create and test biofuels.

Credit: Laurent Brechenmacher, University of Missouri

In addition to conducting the teacher workshops, the researchers have recruited and mentored undergraduate students in plant science research. Through the Freshman Research in Plant Science program, faculty mentors invite first-year students to work in their labs for 8 to 12 hours per week during the academic year. The students also attend weekly meetings led by a senior graduate student who engages them in discussions, presentations and other activities designed to enhance their experiences with plant science research.

The MU biochemistry professor has led groundbreaking research on the soybean

A conversation with: Gary Stacey (Click here to the original story)

BY HUDSON KYLE (Vox magazine)

Much of Gary Stacey’s research hinges on the soybean. The bean is Missouri’s No. 1 crop and the No. 1 vegetable oil in the U.S. Photograph by Harry Katz.

According to a Michigan State study, only 28 percent of adults in the U.S. are considered literate when it comes to science. American students are falling further behind their international peers in mathematics and science testing.

Gary Stacey, an biochemistry professor at MU, says it’s odd that technology is developing at a rapid pace and the world is becoming so much more technologically engaged, yet in many ways, we’re doing a poor job educating people and preparing them for this change.

Stacey and a team from the MU Legume-Microbe Interactions Laboratory hold an annual professional development workshop for Missouri high school science teachers. The workshop offers both the practical knowledge and the physical tools needed to conduct plant experiments in the classroom.

Stacey has led groundbreaking research of the soybean, Missouri’s top cash crop, in his lab, one of the largest on MU’s campus. His research has helped explain what makes the soybean more resilient to adverse growing conditions and contributed to the completion of the soybean genome sequence, which determines an organism’s DNA makeup.

How did you first discover your passion and knack for science?

I think one of the key characteristics of a scientist is curiosity. My dad tells me that when I was a little kid, he used to take me fishing. Instead of fishing, I would be turning over all the rocks. I just wanted to know what was underneath the rocks. Basically, what I’m doing now is turning over rocks, just in a different way. I think a key characteristic is curiosity. If you’re curious, you find ways to maintain that. Science does that for me.

Much of your research involves the soybean. Explain the plant’s significance.

It shouldn’t be hard to understand the importance of the soybean. In most of the Midwest, corn is the No. 1 crop. In Missouri, the soybean is the No. 1 crop. It’s the No. 1 crop in value. It’s the No. 1 crop in acreage. So it’s a very, very important crop.

What do you think the consequences will be if the U.S. science literacy rate continues to decrease

When you talk about the three countries that are fastest growing, you’re talking about Brazil, India and China. Huge investments are taking place there. Especially the Chinese are making huge investments in education, and it’s having an impact.

The fact that we’re an economic power feeds into the fact that we’re a military power, which feeds into the fact that we have geopolitical influence. If we lose our economic power by falling behind, which we already are, then that will affect our military stature and will also undermine our geopolitical influence. That will probably create instability in the world.

 

What is the goal of your teacher-training workshop?

 

Our major objective is to try to get more plant science-related experiments and teaching into the classroom. So often in the classroom, they’ll use an environmental example, or they’ll use a human medical example. That’s all well and good, but we would like to see more plant science being taught. The other objective of these workshops is that I engage my post-doctoral associates. They’re in the laboratory. They’re doing the experiments. But most of them have never had experience in trying to teach clientele like high school teachers.

We’re also able to buy supplies, so the teachers who participate in our workshop actually go home with a big box of stuff. We even get them a light stand they can put in their classroom to grow plants. We provide them with potting materials and seeds and everything they need. It’s all provided for them.

What drives you to give back to the community in the form of this workshop?

It’s fun. You only get one go-around. I wish I realized when I was younger that basically you get one bite at the apple. The other thing is that humans are social animals. You really find that your most enjoyment is dealing with other people. So if you can interact with other people and feel good about what you’ve done, it’s just fun. That’s really what it’s all about. I’m talking about the fun that makes you feel good about yourself.

Missouri just doesn’t invest in schools the way it should. Here in Columbia, a lot of our students until recently were being educated in trailers. I just don’t understand why we don’t make more of an investment.

Contact Information:
Gary Stacey, Ph.D.

Stock Solution number	Element	Final Molarity (μM)	Form	Mol.Wt.	Gram/liter	Molarity of Stock Solution
A	Ca	1000	CaCl2·2H20	147.03	294.1	2.0
B	P	500	KH2P04	136.09	136.1	1.0
C	Fe	10	Fe-Citrate	335.04	6.7	0.02
D	Mg	250	MgSO4·7H20	246.5	123.3	0.5
	K	1500	K2SO4	174.06	87.0	0.5
	S	500
	Mn	1	MnSO4·H20	169.02	0.338	0.002
	B	2	H3BO4	61.84	0.247	0.004
	Zn	0.5	ZnSO4·7H20	287.56	0.288	0.001
	Cu	0.2	CuSO4·5H20	249.69	0.100	0.004
	Co	0.1	CoSO4·7H20	281.12	0.056	0.0002
	Mo	0.1	Na2MoO4·2H20	241.98	0.048	0.0002

Supplies

For extraction of 1 litre of induced Bradyrhizobium japonicum culture requires:

40    grams               XAD-2 Resin (Alltech, XAD-1180 20/60 mesh)

20 ml                      methanol (added to culture)

40 ml                      methanol (elution solvent from resin)

30 ml                      acetone (elution solvent from resin)

2 ml                        18% acetonitrile solution

1                                                        funnel fitted with brass screen

1                                                        2 litre flask

1                                                        250 ml boiling flask

1                            47 mm Whatman #1 filter paper circle

Equipment

Shaker

Flow Cabinet

Fume Hood

Rotary Evaporator

Thermolyne Tube Vortexer

2-litre vacuum filtration system with fritted glass base

Extraction Procedure

1.      Prepare a 1-litre Bradyrhizobium japonicum culture that has been induced with 5 µM of genistein. Grow culture to OD600 of approximately 0.2 to 0.4 .

2.      Before using XAD resins, they must be first conditioned. Place resin in the funnel of the vacuum filtration unit. For each 40 grams of XAD resin, rinse with two washings of 20 ml of acetone, followed by conditioning with 20 ml of methanol, and finally two washings of water (20 ml). Do not use vacuum during each wash. Wash for 1 minute, and then apply vacuum to remove the solvents or water into the collection flask. Allow all water to be removed.

3.      To the culture, add 2% of methanol; for a 1-litre culture use 20 ml of methanol.

4.      Weigh 40 grams of resin and add to flask containing the culture; note that you do not have to centrifuge culture to remove cell pellet.

5.      Place culture flask and resin on shaker and shake overnight (at least 6 hours) at 150 rpm.

6.      After shaking culture and resin, separate culture and resin by pouring the culture through a funnel fitted with a coarse brass mesh. The mesh should be fine enough to allow the culture to flow quickly through, but will filter out the resin beads without any loss. You can fit the funnel over a 2-litre flask to collect the culture. Use water to wash remaining resin beads into funnel from culture flask, and then wash the resin beads that you have collected in the funnel with 1 litre of water.

7.      Fit the fritted glass base of vacuum filtration system with the Whatman #1 paper disk. The purpose of the disk is to collect the beads and to keep  the solvents with the beads until the vacuum is applied. Only when the vacuum is applied should the solvents pass through the glass frit base into the collection flask.

8.      Transfer the resin beads to the funnel on the vacuum filtration system. Use a water bottle to wash all the resin beads into the filter funnel. Apply vacuum to remove water from beads. Discard water collected in flask and rinse flask with methanol to remove water traces.

9.      Wash beads with 40 ml of methanol. Let the methanol wash the beads for 1 minute before applying vacuum. Apply vacuum to system, and collect methanol in flask. After all methanol has been collected, remove vacuum from system.

10.   Wash beads with 30 ml of acetone. As with methanol, wash the beads with the acetone for 1 minute, after which you can apply vacuum to pull the acetone through the filter paper and glass base into the flask. At this time both the methanol and the acetone filtrate are in the flask.

11.  Transfer filtrate to a 250 ml boiling flask and place on a rotary evaporator with a water bath temperature of 45°C and a speed of 125 rpm. Evaporate solvents until flask is dry.

12.  Add 2 ml of 18% acetonitrile to flask and wash the walls of the flask using a vortexer to ensure all residues are dissolved in the solution.

13.  Collect the solution and place in micro centrifuge tube, and spin at 10,000 rpm to sediment particulates. Label and store at -20°C until further analysis by HPLC.

HPLC Analysis

We  have analysed the Nod Factor extract using a Waters HPLC system consisting of the following components;

i) 2 model 510 HPLC pumps

ii) Waters 710 WISP automatic sample injector

iii)Vydac 5µM 300 A c18 column 218TP54  4.6 x 250 mm using a column pre-filter

iv) Waters 410 UV detector at 210 nm

Flow rate: 1.0 ml/min

Solvent: Initial 18:82 acetonitrile:water  gradient as follows;

10-30 minutes 18% to 60% acetonitrile

30-35 minutes 60% to 100% acetonitrile

35-40 minutes 100% to 18% acetonitrile

40-45 minutes 18% acetonitrile

Run Time: 45 minutes

Nominal Elution Time for Nod Factor:  30.75-30.95 minutes

Retention time is consistent with Nod Factor standards that were injected

50 µl of samples are injected.

1. DNase treatment of total RNAs

On ice, mix the following:

*         RNAs                                        10ug (not more than 29.5 ul)

*         10 * RTase buffer                       5ul

*         RQ1 DNase                               2ul

*         H2O                                           up to 36.5ul

Mix well, spin down and incubate at 37C for 15min.

Inactive the DNase at 65C for 10min, then transfer the tubes on ice.

2. RTase (Synthesis of the first strand cDNAs)

On ice, add the following to the DNase-treated RNAs:

*         0.1M DTT                                5ul

*         10mM dNTPs                         5ul

*         0.5ug/ul oligo-dT                    2ul

*         MMLV-RTase                        1ul

*         RNase-inhibitor                      0.5ul

Incubate the tubes 1 hour at 37C then 2min at 92C.

For long storage, store the cDNAs at -80C, otherwise at -20C.

3. PCR

Perform the PCR by using 1 ul of cDNAs for a 50 ul PCR reaction.

Based on Inoue et al (1990), Gene, 96:23-28, with modifications.

1. Culture cells (DH5a in my case) on LB agar plate at 37oC overnight.
2. Pick up 10 -12 large colonies and culture in 250ml SOB in a 1L flask, 19oC with vigorous shaking to OD600=0.5 (normally it takes 24-36hrs)
3. Place the flask in ice for 10 min.
4. Pelleting the cell by spining at 4000rpm for 10 min at 4oC.
5. Gently resuspend the cell in 80ml ice-cold TB and store on ice for 10 min.
6. Spin at 4000rpm for 10 min at 4oC
7. Gently resuspend the pellet in 20ml ice-cold TB and 1.4ml DMSO (the DMSO needs to be stored at -20oC o/n before use).
8. Aliquote the cell to 50 to 500ul for transformation or store at  -70oC

   *         Note: The E. coli cells prepared this way are normally 100 to 1000 times more efficient than normal calcium method, so do not plate too dense!

• SOB solution:

  *         0.5% yeast extract
  *         2% tryptone
  *         10mM NaCl
  *         2.5mM KCl
  *         10mM MgCl2
  *         10mM MgSO4
  *         Dissolve in nanopure water and autoclave to sterilize.

• TB solution:
  *         10mM PIPES
  *         15mM CaCl2
  *         250mM KCl
  *         Dissolve in nanopure water and adjust pH to 6.7 with KOH or
  HCl and then add MnCl2 to 55mM, and adjust to final volume. Sterilize by
  filtration with 0.45um filter and store at 4oC

1.      50-100mg leaf tissue in 1.5 ml eppendorf tube (1 cotyledon for
PCR only)

2.      Prepare fresh microprep buffer, RT

3.      Add 200ul buffer, grind tissue (rinse pestle with water between
samples). Add another 55ul buffer, shake entire rack by hand.

4.      65C, 30-120min

5.      Fill the tube with chloroform, mix well (shaking up and down
50-100 times)

6.      10000 rpm, 5min

7.      Pipet off aqueous phase (~0.5ml), add 1X volume of cold
isopropanol, invert tube repeatedly until DNA precipitates.

8.      Immediately spin at 10000rpm for 5min (No more)

9.      Wash pellet with 70% ethanol

10.  Dry

11.  Resuspend in 50ul TE at 65C for 15min

12.  Spin 10min at 10000rpm, store at -20C.

13.  1ul for PCR, 15-25ul for southern blot (5-10ug DNA, 15-20 units
enzyme) (If 1 cotyledon was used, 5ul for PCR)

DNA extraction buffer(pH 7.5)

50ml

100ml

Final concentration

Sorbitol (MW 182.2)

3.19g

6.38g

0.35M

Tris-base (1M)

5ml

10ml

0.1M

EDTA (0.5M)

0.5ml

1ml

5mM

Nuclei lysis buffer:

15ml

30ml

Final concentration

Tris (1M)

10ml

20ml

0.2M

EDTA (0.5M)

5ml

10ml

0.05M

NaCl

5.84g

11.68g

2M

CTAB

1g

2g

2%

Sarkosyl: 5% (w/v)

Microprep buffer:

DNA extraction buffer

25ml

15ml

10ml

Nuclei lysis buffer

25ml

15ml

10ml

5% sarkosyl

10ml

6ml

4ml

Sodium bisulfite

0.2g

0.12g

0.08g

1.        Stock solution

(1)    5×MOPS

ddH2O                                          400ml

3M NaOAc (pH7.0)                      6.67ml

0.5M EDTA (pH8.0)                         5ml

MOPS                                           10.4g

NaOH                                           ~0.875g to pH7.0

Add ddH2O to 500ml, add 500ul DEPC

Set overnight, autoclave for 15min.

(2)    0.5M Na3PO4 (pH7.2)

ddH2O                                            1800ml

NaH2PO4·H2O                               43.6g

Na2HPO4·7H2O                         183.3g

Add ddH2O to 2l, autoclave for 15min.

(3)    10×SSC

ddH2O                                              1800ml

NaCl                                                  175g

Trisodium citrate                                88.2g

Adjust pH to 7.0 with HCl, add ddH2O to 2l, add 2ml DEPC

Set overnight, autoclave for 15min.

(4)    DEPC ddH2O

(5)    14% SDS

2.        Electrophoresis

(1)    Formaldehyde gel ( Pretreat comb and plate with ethanol and Rnase away)

50ml             100ml               200ml           250ml

Agarose             0.5g              1.0g                 2.0g               2.5g

5×MOPS          10ml             20ml                 40ml              50ml

DEPC H2O       38ml             77.5ml              155ml           194ml

Melt

65C H2O bath 10min

Add 37% Formaldehyde  2.7ml           5.4ml                10.8ml          13.5ml

Pour gently

Set 15-20min

(2)    Running buffer (1×MOPS )

250ml             600ml             1000ml

DEPC H2O        200ml             480ml              800ml

5×MOPS             50ml             120ml              200ml

(3)    Sample preparation (30ul)

Formamide                                               15ul

5×MOPS                                                    6ul

formaldehyde                                            4.8ul

RNA                                                          10ug ( less than 4.2ul)

DEPC H2O                                                 add to 30ul

65C 10min, put on ice, add 3ul 10×loading buffer.

(4)    Electrophoresis ( Pretreat tank and cylinder with Sparkleen)

Pre- electrophoresis gel at 5V/cm for 5min

Load samples

Run gel at 4V/cm for 1-2h.

3.        Transfer of RNA from Gel to Membrane

Wash gel with DEPC H2O  3 times ( 20min RT H2O, 10min 65C H2O, 20min RT H2O)

Wash with 10×SSC for 30min

Vacuum blot 90min at 5in pressure, using 10×SSC as transfer buffer

Rinse with 2×SSC for 5min

80C bake 30min.

4.        Hybridization

(1)    (Pre)hybridization solution       50ml ( 30ml for prehybridization, 20ml for hybridization)

0.5M Na3PO4 (pH7.2)                 25ml

14% SDS                                     25ml

(2)    Probe labeling

Probe                                              100ng

ddH2O                                             add to 30ul

boil 5min, put on ice

5×labeling mix                                10ul

d(C,T,G)TP mix (1.5mM)                 2ul

BSA(10mg/ml)                                  2ul

Klenow(5u/ul)                                   1ul

P32-dATP                                         5ul

RT 2hrs, add 2ul 0.5M EDTA, boil 10min, G-50 micro column clean, use for hybridization.

(3)    Prehybridization (65C) for more than 2 hrs( while probe labeling), hybridization (65C) overnight.

(4)    Wash

14% SDS                                               89ml

Na3PO4                                                  10ml

ddH2O                                                    151ml

wash for 2 times at 65C, use 125ml wash solution each time.

(5)    Strip

10×SSC                                                    10ml

14% SDS                                                    35ml

ddH2O                                                     955ml

wash for 2 times in boiling solution, use 500ml solution each time.