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Lab notebook

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1.Protocol

We used 4 different strains

• NifB～hesA(9669 bp) Paenibacillus polymyxa ATCC 15970
• NifVSU(3324 bp) Rahnella aquatilis NBRC 105701
• pheDH (1189 bp) Bacillus badius NBRC 15713
• gluDH(1437 bp) Bacillus licheniformis NBRC 12200

We used the same marker for the result of electrophoresis.

• pMW-f: cccggggatc ctctagagtc gacct


• pMW-r: gagctcgaat tcgtaatcat ggtca


• nifVSU-f(infusion): tgaccatgat tacgaattcg agctc GTACGACATC ACCCGAACCG


• nifVSU-r(infusion): aggtcgactc tagaggatcc ccggg TCAGCCTCCC TGAGCCTGAA


• gluDH-f:
  tcaggctcagggaggctgacccgggAGAAAGGGAGCTGAAAAAAGATG
• gluDH-r:
  tgcatgcctgcaggtcgactctagaTTAAACGACTCCCTGAGCGA


• pheDH-f :
  tcaggctcagggaggctgacccgggGGCTAAATAAAAGCGTTCAA


• pheDH-r:
  tgcatgcctgcaggtcgactctagaTTAGTTGCGAATATCCCAT


• pMWnifVSU-f:
  tctagagtcg acctgcaggc atgca


• pMWnifVSU-r: cccgggtcag cctccctgag cctga



※Capital letter is complementary to the ends of the linearized vector notebook. We took over the last years project. So we want to check last year’s notebook before reading this year’s notebook.

Last year's notebook

LB medium




Materials	Volume
NaCl	10 g
yeast extract	5.0g
typtone	10g
dH2O	1L
pH 6.8～7.2




LB agar medium




Materials	Volume
NaCl	10 g
yeast extract	5.0g
typtone	10 g
Agarose	1.5 %
dH2O	1L
pH 6.8～7.2




Glutamic free medium　preparation Glutamic free medium contain following


b>Glutamic free medium　preparation
Glutamic free medium contain following


Na2HPO4,      　　　      10.4g
 
KH2PO4,      　　　        3.4g

CaCl2·2H2O,      　　　    26mg

30 mg MgSO4,      　　　   30mg

MnSO4,      　　　        0.3mg

Ferric citrate,      　　　36mg
 
Na2MoO4·2H2O      　　　   7.6mg

p-aminobenzoic acid,      10μg

biotin      　　　         5μg

glucose                      4g

distilled water      　　　  1L 




Nitrogen deficient medium　preparation Nitrogen deficient medium contain following Add Glutamate to Nitrogen Free medium by 5 mM.




PCR or colony PCR




Mix PCR solutions and run the PCR machine in a program which is detailed below.




PCR Solution	Primer-F 10µM	template	Primer-R 10 µM	2mM dNTPs	2 x PCR Buffer	KOD Fx Neo DNA Polymerase	DW	Total
Volume (µL)	1.5	x	1.5	10	25	1	11	50+x




PCR
Cycle 1:(1 x) Step 1 94 ℃ for 2:00
Cycle 2:(35 x) Step１ 98 ℃ for 0:10 Step 2 68 ℃ for 08:00(pMWnifVSU)
for 2:00(pheDH,gluDH)

Cycle 3:(1 x) Step 1 4 ℃ for ∞




Electrophoresis

・Prepare agarose gel

1. Measure 0.2 g of agarose (1-2 %) to 20 mL of TAE in a beaker
2. Microwave until all agarose has dissolved
3. Allow agarose solution to cool down
4. Pour solution into gel tray.
5. Leave until gel has solidified then remove the com
6. Insert the gel into the electrophoresis chamber with the wells closest to the negative (black) electrode.
7. Gradually add 1xTBE (Tris-Borate-EDTA; electrophoresis buffer) to the chamber until the buffer just covers the top of the gel.
8. Add 2 µl of 6 x loading buffer to 5 µl of each sample.
9. Add 5 µl of each sample with 2 µl of 10 x loading buffer and 0.5~5 kb marker .
10. Start electrophoresis at 100 V.
11. Stop at appropriate time.
12. Put the gel in the container which is filled with EtBr




Cloning

We used in-fusion cloning

5X In-Fusion HD Enzyme Premix	２µl
Linearized vector	4 µl
PCR fragment	4 µl
total volume	10 µl

• We refined PCR mixture by GFX　pulification kit.
• Set up the in-Fusion cloning reaction.



• Incubate the reaction for 15 min at 50 °C, then place on ice.
• store the cloning reaction at refrigerator(-20 ℃) over night.



Transformation

1. Thaw competent cells(JM109) on ice
2. Add plasmid DNA (1 µl) to competent cells. leave them on ice for 30 minutes
3. Keep them in heating block(42 ℃) for 45 seconds, then cool on ice for 1 minutes
4. Add 500 µl SOC liquid culture medium, then incubate with shaking for 1hour at 37 °C
5. Seed cells onto LB+ampicillin agar plate. Incubate for 24 hours at 37 ℃



Gel purification




We used illustra GFX PCR DNA and Gel Band Purification Kit


１.Sample Capture
• Using a clean scalpel, long wavelength (365 nm) ultraviolet light and minimal exposure time, cut out an agarose band containing the sample of interest.
• Add 10 µl Capture buffer type 3 for each 10 mg of gel slice, for example, add 300 µl Capture buffer type 3 to each 300 mg gel slice.
• Mix by inversion and incubate at 60 °C for 15–30 minutes until the agarose is completely dissolved. Mix by inversion every 3 minutes.
• For each purification that is to be performed, place one GFX MicroSpin column into one Collection tube.

２. Sample Binding
• Centrifuge Capture buffer type 3- sample mix briefly to collect the liquid at the bottom of the tube.
• Transfer up to 800 µl Capture buffer type 3- sample mix onto the assembled GFX MicroSpin column and Collection tube.
• Incubate at room temperature for 1 minute.
• Spin the assembled column and Collection tube at 16,000 × g for 30 seconds.
• Discard the flow through by emptying the Collection tube. Place the GFX MicroSpin column back inside the Collection tube.
• f. Repeat Sample Binding steps b. to e. as necessary until all sample is loaded.

３. Wash & Dry
• a. Add 500 µl Wash buffer type 1 to the GFX MicroSpin column.
• b. Spin the assembled column and Collection tube at 16,000 × g for 30 seconds
• c. Discard the Collection tube and transfer the GFX MicroSpin column to a fresh DNase-free 1.5 ml microcentrifuge tube (supplied by user).

４.Elution

1. a. Add 10–50 µl Elution buffer type 4 or type 6 to the center of the membrane in the assembled GFX MicroSpin column and sample Collection tube.
2. b. Incubate the assembled GFX MicroSpin column and sample Collection tube at room temperature for 1 minute.
3. c. Spin the assembled column and sample Collection tube at 16,000 × g for 1 minute to recover the purified DNA.
4. d. Proceed to downstream application. Store the purified DNA at -20 °C.



Purification of plasmid DNA

We used QIAprep Spin Miniprep Kit

1. Resuspend pelleted bacterial cells in 250 μl Buffer P1 and transfer to a microcentrifuge tube.
2. Add 250 μl Buffer P2 and gently invert the tube 4–6 times to mix.
3. Add 350 μl Buffer N3 and invert the tube immediately but gently 4–6 times.
4. Centrifuge for 10 min at 13,000 rpm (~17,900 x g) in a table-top microcentrifuge.
5. Apply the supernatants from step 4 to the QIAprep spin column by decanting or pipetting.
6. Centrifuge for 30–60 s. Discard the flow-through.
7. (Optional): Wash the QIAprep spin column by adding 0.5 ml Buffer PB and centrifuging for 30–60 s. Discard the flow-through.
8. Wash QIAprep spin column by adding 0.75 ml Buffer PE and centrifuging for 30–60 s.
9. Discard the flow-through, and centrifuge for an additional 1 min to remove residual wash buffer.
10. Place the QIAprep column in a clean 1.5 ml microcentrifuge tube. To elute DNA, add 50 μl Buffer EB (10 mM Tris・Cl, pH 8.5) to the center of each QIAprep spin column, let stand for 1 min, and centrifuge for 1 min.

※pre-heat Buffer EB (or water) to 70 °C prior to eluting DNA

Restriction digests

We used sac1 and Xma1 as restriction enzyme.

1. Add 68 ul of DNA to be digested into a 1.5 ml microcentrifuge tube.
2. Add 10 ul of 10x buffer.
3. Add 20 ul of pSMW218 or PCR product.
4. Add 2.0 ul of Sac1.
5. Incubate the restriction digest at 37 ℃ for 5 hour.



Purification of DNA from an enzymatic reaction by GFX　pulification kit.

1. Add 68 ul of DNA to be digested into a 1.5 ml microcentrifuge tube.
2. Add 10 ul of 10x buffer.
3. Add 20 ul of pSMW218 or PCR product.
4. Add 2.0 ul of Xma1.
5. Incubate the restriction digest at 37 ℃ for overnight.



Purification of DNA from an enzymatic reaction by GFX pulification kit.

Purification of DNA from PCR mixture

• a.Add 500 μL of capture buffer type 3 to 100 μL of sample.
• b.Pipetting
• c.Centrifuge for 1 min at 15,000 rpm (~17,900 x g).
• d.Discard the flow through
• e.Add 500 μL　of wash buffer.
• f. Centrifuge for 1 min at 15,000 rpm (~17,900 x g)



Indophenol




Day1

• a. The samples are planted in 5 ml of antibiotic LB medium.
• b.Incubate 24 h at 30 ℃



Day2

• c. 180 µL of incubated LB medium and samples are added to nitrogen free medium.
• d. Add Glutamate as nitrogen source(Nitrogen deficient medium)
• e .Add appropriate antibiotics.
• f. If sample has pMW plasmid, Add IPTG to activate Lac promoter. Add MSX if necessary.
• e. Fill Nitrogen into Nitrogen deficient medium.
• g.Incubate at 30 ℃



Day3~5

• h.Add indophenol reagent.
• I.Wait 30 minitues.
• J .Measure abs 600

Indophenol reagent

Materials	Volume (µl)
Phenol/EtOH	40
Sodium nitroprusside/H2O	40
Sodium hypochlorite	100






2.Laboratory notebook



5/21

LB medium preparation

LB agar medium preparation(Anitibiotics:Amp,Km,Amp+Km)




5/22

LB agar medium preparation

LB agar medium preparation




5/24

Transformation

Sample Name	Sample Volume (μl)	Competent cell(E.coli)/(μl)	Medium
pHYnifB,H,D,K,N,X,hesA	1	20	SOC Medium
pMWnifVSU	1	20	SOC Medium
pHYnifB,H,D,K,N,X,hesA+pHYnifVSU	1	20	SOC Medium
pHY	1	20	SOC Medium
pHYpMW	0.5+0.5	20	SOC Medium




5/25

Preculture

Transformant is planted in 5 ml of LB medium and streaked to antibiotic LB agar medium
~Failed~




5/30

Preculture

Name	medium	Antibiotics
E.coli(pHY)	LB	Amp
E.coli(pHYnifB,H,D,K,N,X,hesA)	LB	Amp
E.coli(pMW)	LB	Km
E.coli(pMWnifB,H,D,K,N,X,hesA)	LB	Km
E.coli(pHYpMW)	LB	Amp,Km
E.coli(pHYnifB,H,D,K,N,X,hesA,pMWnifVSU)	LB	Amp,Km




5/31

Plasmid Extraction

Sample: Precultured sample




6/1

Electrophresis

Lane	sample/(µl)
1.pHY	5
2.pHYnif	5
3.pMW	5
4.pMWnif	5
5.pHYpMW	5
6.pHYnifB,H,D,K,N,X,hesA,pMWnifVSU





6/4

Electrophresis

Lane	sample/(µl)
1.pMW	5
2.pMW	5
3.pMWnifVSU	5
4.pMWnifVSU	5
5.pHYnifB,H,D,K,N,X,hesA,pMWnifVSU	5





6/4

LB medium preparation




6/6

Transformation

Sample Name	Sample Volume (µl)	Competent Cells(JM109)/(µl)	Medium
pHY	1	20	SOC Medium
pHY	1	20	SOC Medium




6/11

LB agar medium preparation(Antibiotics: Amp)




6/14

Transformation

Sample Name	Sample Volume (µl)	Competent Cells(JM109)/(µl)	Medium
pHY	1	20	SOC Medium
pHY	1	20	SOC Medium




6/18

LB agar prepararation




6/25

Colony PCR

Templates/(µl)	Primers/(µl)	Primer's name	MilliQ/(µl)	PreMix/(µl)	Total/(µl)
pMW	1.5	pMW	1	50	51
pMW	1.5	pMW	1	50	51
Rahenella aquatilis	1.5	nifVSU	1	50	51
Rahenella aquatilis	1.5	nifVSU	1	50	51
Rahenella aquatilis	1.5	nifVSU	1	50	51
Rahenella aquatilis	1.5	nifVSU	1	50	51




6/26

Electrophresis

Lane	sample/µl)
1..pMW	5
2..pMW	5
3.nifVSU for restriction enzyme	5
4.nifVSU for restriction enzyme	5
5.nifVSU for infusion	5
6.nifVSU for infusion	5





6/28

Electrophresis

Lane	sample/(µl)
1.pMW	5
2.pMW	5
3.nifVSU for restriction enzyme	5
4.nifVSU for restriction enzyme	5





7/2

Purification of DNA from PCR mixture

Sample:
June26,sample5
June 28,sample2
June 28,sample4





7/3

Infusion Cloning

pMW and nifVSU




7/3

Electrophresis

Lane	sample/(µl)
1.nifVSU	5
2.pMW	5
3.pHY	5
4.none	5
5.nifVSU	5
6.pMW	5




7/4

Transformation

Sample Name	Sample Volume (µl)	Competent Cells(JM109)/(µl)	Medium
pMW	1	20	SOC Medium
pMWnifVSU	1	20	SOC Medium




7/5

pMW streaked to antibiotic LB agar medium




7/6

Km,amp, stock tube preparation




7/9

Anitibiotick check




7/10

Transformation

Sample Name	Sample Volume (µl)	Competent Cells(JM109)/(µl)	Medium
pMW	1	20	SOC Medium
pMWnifVSU	1	20	SOC Medium




7/11

Colony PCR

Templates/(µl)	Primers/(µl)	Primer's name	MilliQ/(µl)	PreMix/(µl)	Total/(µl)
Rahnella aquatillis	1.5	nifVSU	1	50	51
pMW	1.5	nifVSU	1	50	51
pMWnifVSU	1.5	nifVSU	1	50	51
pMWnifVSU	1.5	nifVSU	1	50	51
pMWnifVSU	1.5	nifVSU	1	50	51
pMWnifVSU	1.5	nifVSU	1	50	51




7/12

Electrophresis

Lane	sample/(µl)
1.NifV,S,U	5
2.NifV,S,U	5
3.NifV,S,U	5
4.NifV,S,U	5
5.NifV,S,U	5
6.NifV,S,U	5




7/12

Preculture for plasmid extraction

Sample:
1.JM109(pMW)
2.Transformant(pMWnifVSU) 1


7/13

Electrophresis

Lane	sample/(µl)
1.pMW	5
2.pMWVSU	5
3.pMW Sac1	5
4.VSU Sac1 pMW	5
5.pMW Xma1	5
6.VSU Xma1 pMW	5




8/9

Transformation

Sample Name	Sample Volume (µl)	Competent Cells(JM109)/(µl)	Medium
pMWpHY	0.5+0.5	20	SOC Medium
pMWnifVSU +pHYnifB,H,D,K,N,X,hesA	0.5+0.5	20	SOC Medium




8/21

Transformation

Sample Name	Sample Volume (µl)	Competent Cells(JM109)/(µl)	Medium
pHY	0.5+0.5	20	SOC Medium
pHYnifB,H,D,K,N,X,hesA,pMW	0.5+0.5	20	SOC Medium
pHY+pMW	0.5+0.5	20




8/22

Preculture for plasmid extraction

Electrophoresis




Lane	sample/(µl)
1.pHYnifB~hesApMWnifVSU	5
2.pHY,pMW	5
3.pHY	5
4.pHYnifB~hesA	5
5.pMW	5
6.pMWnifVSU	5





8/30

Colony PCR

Templates/(µl)	Primers/(µl)	Primer's name	MilliQ/(µl)	PreMix/(µl)	Total/(µl)
pMWnifVSU	1.5	pMWnifVSU	1	50	51
pMWnifVSU	1.5	pMWnifVSU	1	50





9/6

Colony PCR

Templates/(µl)	Primers/(µl)	Primer's name	MilliQ/(µl)	PreMix/(µl)	Total/(µl)
Bacilllus lichenformis	1.5	gluDH	1	50	51
Bacilllus lichenformis	1.5	gluDH	1	50	51
Bacilllus lichenformis	1.5	gluDH	1	50	51
Bacilllus lichenformis	1.5	gluDH	1	50	51
pMWVSU extracted from transformant made July 10	1.5	gluDH	1	50	51
pMWVSU extracted from transformant made July 10	1.5	gluDH	1	50




9/10

Gel extraction

Sample: September 6 Sample2




9/11

Electrophoresis

sample:purified nifVSU





Transformation

Sample Name	Sample Volume (µl)	Competent Cells(JM109)/(µl)	Medium
PMWnifVSU+pheDH	0.5+0.5	20	SOC Medium
PMWnifVSU+gluDH	0.5+0.5	20	SOC Medium
pMWnifVSU+gluDH	0.5+0.5	20	SOC Medium

9/12

Preculture of Transformant

9/13

Plasmid extraction

1.pMWpheDH
2.pMWpheDH+pHYnif
3.pMW
4.pMW+pHY





9/14

Restriction enzyme digest




9/17

Lane	sample/(µl)
1.gluDH	5
2.gluDH	5
3.pMWnifVSU	5
4.pMWnifVSU	5




9/20

Colony PCR

Templates/(µl)	Primers/(µl)	Primer's name	MilliQ/µl)	PreMix/(µl)	Total/(µl)
Bacilllus lichenformis	1.5	gluDH	1	50	51
Bacilllus lichenformis	1.5	gluDH/i>	1	50	51
JM109(pMWnifVSU)	1.5	pMWnifVSU	1	50	51
JM109(pMWnifVSU)	1.5	pMWnifVSU	1	50	51





9/21

Electrophresis

Lane	sample/(µl)
1.gluDH	5
2.gluDH	5
3.pMWnifVSU	5
4.none	5
5.pMWnifVSU	5
6.none	5





9/25

DNA extraction from Agarose gel

Sample:
1.pMWnifVSU
2.pMWnifVSU





Infusion cloning

Sample:pMWnifVSU+gluDH




10/8

transformation

Sample Name	Sample Volume (µl)	Competent Cells/(µl)	Medium
pMWnifVSUgluDH	1.0	20	SOC
pHYnifB~hesA+pMWnifVSUgluDH	0.5,0.5	20	SOC
pMW(positive control)	1.0	20	SOC




10/10

Preculture for plasmid extraction

Name	medium	Antibiotics
E.coli(pMWnifVSUgluDH)	LB	Km
E.coli(pHYnifB~hesA,pMWnifVSUgluDH)	LB	Amp,Km






Indophenol method




5/23

Nitrogen free medium A,B,C and D preparation




6/19

Preculture for indophenol

Name	medium	Antibiotics
Paenibacillus polymyxa	LB	none
E.coli	LB	none




6/20

Inoculation

Sample Name	glutamate(mM)	Antibiotics	100mM IPTG(µl)	50mM MSX(µl)	Nitrgen free medium(ml)
Paenibacillus polymyxa	25	none	0	0	2.5
E.coli	25	none	２５0	0	2.5
Paenibacillus polymyxa	25	none	0	0	2.5
E.coli	25	none	0	0	2.5




6/20

Inoculation

Sample Name	Phenol/EtOH(µl)	Sodium nitroprusside/H2O(µl)	Sodium hypochlorite(µl)
Paenibacillus polymyxa	40	40	100
E.coli	40	40	100
Paenibacillus polymyxa	40	40	100
E.coli	40	40	100




6/21

Indophenol(24h)

Kasahara

Sample Name	Phenol/EtOH(µl)	Sodium nitroprusside/H2O(µl)	Sodium hypochlorite(µl)
Paenibacillus polymyxa	40	40	100
E.coli	40	40	100
Paenibacillus polymyxa	40	40	100
i>E.coli	40	40




6/22

Indophenol(48h)

Kasahara

Sample Name	Phenol/EtOH(µl)	Sodium nitroprusside/H2O(µl)	Sodium hypochlorite(µl)
Paenibacillus polymyxa	40	40	100
E.coli	40	40	100
Paenibacillus polymyxa	40	40	100
E.coli	40	40




9/2

Preculture for indophenol

Sample Name	medium	Antibiotics
E.coli(pHYnifB~hesA)	LB	Amp
E.coli(pHYnifB~hesA+pMWnifVSU)	LB	Amp+Km
E.coli(pHY)	LB	Amp
E.coli(pHYnifB~hesA+pMW)	LB	Amp+Km
E.coli(pHY+pMW)	LB	Amp+Km
Paenibacillus polymyxa	LB	none




9/3

Inoculation

Sample Name	glutamate(mM)	Antibiotics	100mM IPTG(µl)	50mM MSX(µl)	Nitrgen free medium(ml)
E.coli(pHYnifB~hesA)48 h	25	Amp	0	0	2.5
E.coli(pHYnifB~hesA)72 h	25	Amp	0	0	2.5
E.coli(pHYnifB~hesA+pMWnifVSU)48h	25	Amp,Km	25	0	2.5
E.coli(pHYnifB~hesA+pMWnifVSU)72 h	25	Amp,Km	25	0	2.5
E.coli(pHY)48 h	25	Amp	0	0	2.5
E.coli(pHY)72 h	25	Amp	0	0	2.5
E.coli(pHYnifB~hesA+pMW)48 h	25	Amp,Km	25	0	2.5
E.coli(pHYnifB~hesA+pMW)72 h	25	Amp,Km	25	0	2.5
E.coli(pHY+pMW)48 h	25	Amp,Km	25	0	2.5
E.coli(pHY+pMW)72 h	25	Amp,Km	25	0	2.5
Paenibacillus polymyxa 48 h	25	none	0	0	2.5
Paenibacillus polymyxa 72 h	25	none	0	0	2.5
Medium only	25	none	0	0	2.5
Medium only	25	none	0	0	2.5
E.coli(pHYnifB~hesA,pMWnifVSU)48 h MSX	25	Amp,Km	25	25	2.5
E.coli(pHYnifB~hesA,pMWnifVSU)72 h MSX	25	Amp,Km	25	25	2.5
E.coli(pHY+pMW)48 h MSX	25	Amp,Km	25	25	2.5
E.coli(pHY+pMW)72 h MSX	25	Amp,Km	25	25	2.5
Paenibacillus polymyxa 48 h MSX	25	none	0	25	2.5
Paenibacillus polymyxa 72 h MSX	25	none	0		2.5




9/5

Indophenol(48 h)

Sample Name	Phenol/EtOH(µl)	Sodium nitroprusside/H2O(µl)	Sodium hypochlorite(µl)
1.E.coli(pHYnifB~hesA)	40	40	100
2.E.coli(pHYnifB~hesA+pMWnifVSU)	40	40	100
3.E.coli(pHY)	40	40	100
4.E.coli(pHYnifB~hesA+pMW)	40	40	100
5.E.coli(pHY+pMW)	40	40	100
6.Paenibacillus polymyxa	40	40	100
7.Medium only	40	40	100





Indophenol(48 h)MSX

Sample Name	Phenol/EtOH(µl)	Sodium nitroprusside/H2O(µl)	Sodium hypochlorite(µl)
1.E.coli(pHYnifB~hesA+pMWnifVSU)	40	40	100
2.E.coli(pHY+pMW)	40	40	100
3.Paenibacillus polymyxa	40	40	100
4.Medium only	40	40	100





9/6

Indophenol(72 h)

Sample Name	Phenol/EtOH(µl)	Sodium nitroprusside/H2O(µl)	Sodium hypochlorite(µl)
1.E.coli(pHYnifB~hesA)	40	40	100
2.E.coli(pHYnifB~hesA+pMWnifVSU)	40	40	100
3.E.coli(pHY)	40	40	100
4.E.coli(pHYnifB~hesA+pMW)	40	40	100
5.E.coli(pHY+pMW)	40	40	100
6.Paenibacillus polymyxa	40	40	100
7.Medium only	40	40	100





Indophenol(72 h)MSX

Sample Name	Phenol/EtOH(µl)	Sodium nitroprusside/H2O(µl)	Sodium hypochlorite(µl)
1.E.coli(pHYnifB~hesA+pMWnifVSU)	40	40	100
2.E.coli(pHY+pMW)	40	40	100
3.Paenibacillus polymyxa	40	40	100
4.Medium only	40	40	100





9/10

Preculture for indophenol

Sample Name	Medium	Antibiotics
E.coli(pHYnifB~hesA)	LB	Amp
E.coli(pHYnifB~hesA+pMWnifVSU)	LB	Amp,Km
E.coli(pHY)	LB	Amp
E.coli(pHYnifB~hesA+pMW)	LB	Amp,Km
E.coli(pHY+pMW)	LB	Aap,Km
Paenibacillus polymyxa	LB	none
Medium only	LB	none




9/11

Inoculation

Sample Name	glutamate(mM)	Antibiotics	100mM IPTG(µl)	50mM MSX(µl)	Nitrgen free medium(ml)
E.coli(pHYnifB~hesA)	25	Amp	0	0	2.5
E.coli(pHYnifB~hesA+pMWnifVSU)	25	Amp,Km	25	0	2.5
E.coli(pHY)	25	Amp	0	0	2.5
E.coli(pHYnifB~hesA+pMW)	25	Amp,Km	25	0	2.5
E.coli(pHY+pMW)	25	Amp,Km	25	0	2.5
Paenibacillus polymyxa	25	none	0	0	2.5
medium only	25	none	0	0	2.5
E.coli(pHYnifB~hesA+pMWnifVSU) MSX	25	Amp,Km	25	25	2.5
E.coli(pHY) MSX	25	Amp	0	25	2.5
E.coli(pHYnifB~hesA+pMW) MSX	25	Amp,Km	25	25	2.5
E.coli(pHY+pMW) MSX	25	Amp,Km	25	25	2.5
MSX	25	none	0	25	2.5




9/14

Indopheol(72h)

Sample Name	Phenol/EtOH(µl)	Sodium nitroprusside/H2O(µl)	Sodium hypochlorite(µl)
1.E.coli(pHYnifB~hesA)	40	40	100
2.E.coli(pHYnifB~hesA+pMWnifVSU)	40	40	100
3.E.coli(pHY)	40	40	100
4.E.coli(pHYnifB~hesA+pMW)	40	40	100
5.E.coli(pHY+pMW)	40	40	100
6.Paenibacillus polymyxa	40	40	100
7.medium only	40	40	100





Indopheol(72h)MSX

Sample Name	Phenol/EtOH(µl)	Sodium nitroprusside/H2O(µl)	Sodium hypochlorite(µl)
1.E.coli(pHYnifB~hesA+pMWnifVSU) MSX	40	40	100
2.E.coli(pHY) MSX	40	40	100
3.E.coli(pHYnifB~hesA+pMW) MSX	40	40	100
4.E.coli(pHY+pMW) MSX	40	40	100
5.Paenibacillus polymyxa MSX	40	40	100





9/24

Preculture for indophenol

Sample Name	medium	Antibiotics
E.coli(pHYnifB~hesA)	LB	Amp
E.coli(pHYnifB~hesA+pMWnifVSU)	LB	Amp,Km
E.coli(pHY)	LB	Amp
E.coli(pHYnifB~hesA+pMW)	LB	Amp,Km
E.coli(pHY+pMW)	LB	Aap,Km
Paenibacillus polymyxa	LB	none
Medium only	LB	none




9/25

Inoculation

Sample Name	glutamate(mM)	Antibiotics	100 mM IPTG(&microl)	50 mM MSX(&microl)	Nitrgen free medium(ml)
E.coli(pHYnifB~hesA)48 h	25	Amp	0	0	2.5
E.coli(pHYnifB~hesA+pMWnifVSU)48h	25	Amp,Km	25	0	2.5
E.coli(pHY)48 h	25	Amp	0	0	2.5
E.coli(pHYnifB~hesA+pMW)48 h	25	Amp,Km	25	0	2.5
E.coli(pHY+pMW)48 h	25	Amp,Km	25	0	2.5
Paenibacillus polymyxa 48 h	25	none	0	0	2.5
Medium only	25	none	0	0	2.5
E.coli(pHYnifB~hesA,pMWnifVSU)48 h MSX	25	Amp,Km	25	25	2.5
E.coli(pHY+pMW)48 h MSX	25	Amp,Km	25	25	2.5




9/28

Indophenol(48h)-1

Sample Name	Phenol/EtOH(&microl)	Sodium nitroprusside/H2O(&microl)	Sodium hypochlorite(&microl)
1.E.coli(pHYnifB~hesA)	40	40	100
2.E.coli(pHYnifB~hesA+pMWnifVSU)	40	40	100
3.E.coli(pHY)	40	40	100
4.E.coli(pHYnifB~hesA+pMW)	40	40	100
5.E.coli(pHY+pMW)	40	40	100
6.Paenibacillus polymyxa	40	40	100
7.Medium only	40	40





Indophenol(48h)MSX-1

Sample Name	Phenol/EtOH(&microl)	Sodium nitroprusside/H2O(&microl)	Sodium hypochlorite(&microl)
1.E.coli(pHYnifB~hesA+pMWnifVSU) MSX	40	40	100
2.E.coli(pHY+pMW)MSX	40	40	100





9/28

Indophenol(48h)-2

Sample Name	Phenol/EtOH(&microl)	Sodium nitroprusside/H2O(&microl)	Sodium hypochlorite(&microl)
1.E.coli(pHYnifB~hesA)	40	40	100
2.E.coli(pHYnifB~hesA+pMWnifVSU)	40	40	100
3.E.coli(pHY)	40	40	100
4.E.coli(pHYnifB~hesA+pMW)	40	40	100
5.E.coli(pHY+pMW)	40	40	100
6.Paenibacillus polymyxa	40	40	100
7.Medium only	40	40





Indophenol(48h)MSX-2

Sample Name	Phenol/EtOH(&microl)	Sodium nitroprusside/H2O(&microl)	Sodium hypochlorite(&microl)
1.E.coli(pHYnifB~hesA+pMWnifVSU) MSX	40	40	100
2.E.coli(pHY+pMW)MSX	40	40	100





9/28

Indophenol(48h)-3

Sample Name	Phenol/EtOH(&microl)	Sodium nitroprusside/H2O(&microl)	Sodium hypochlorite(&microl)
1.E.coli(pHYnifB~hesA)	40	40	100
2.E.coli(pHYnifB~hesA+pMWnifVSU)	40	40	100
3.E.coli(pHY)	40	40	100
4.E.coli(pHYnifB~hesA+pMW)	40	40	100
5.E.coli(pHY+pMW)	40	40	100
6.Paenibacillus polymyxa	40	40	100
7.Medium only	40	40





Indophenol(48h)MSX-3

Sample Name	Phenol/EtOH(&microl)	Sodium nitroprusside/H2O(&microl)	Sodium hypochlorite(&microl)
1.E.coli(pHYnifB~hesA+pMWnifVSU) MSX	40	40	100
2.E.coli(pHY+pMW)MSX	40	40	100





9/28

Indophenol(48h)-4

Sample Name	Phenol/EtOH(&microl)	Sodium nitroprusside/H2O(&microl)	Sodium hypochlorite(&microl)
1.E.coli(pHYnifB~hesA)	40	40	100
2.E.coli(pHYnifB~hesA+pMWnifVSU)	40	40	100
3.E.coli(pHY)	40	40	100
4.E.coli(pHYnifB~hesA+pMW)	40	40	100
5.E.coli(pHY+pMW)	40	40	100
6.Paenibacillus polymyxa	40	40	100
7.Medium only	40	40





Indophenol(48h)MSX-4

Sample Name	Phenol/EtOH(&microl)	Sodium nitroprusside/H2O(&microl)	Sodium hypochlorite(&microl)
1.E.coli(pHYnifB~hesA+pMWnifVSU) MSX	40	40	100
2.E.coli(pHY+pMW)MSX	40	40	100





10/04

Detection of glutamate




10/8

transformation

Sample

1.pMWnifVSU+gluDH

2.pHYnifB~hesA,pMWnifVSU
+gluDH

3.pMW

10/9

preculture

Miniprep

Sample:transformant(10/8)

10/11

Miniprep

Sample:Precultured E.coli

Electrophoresis




Lane	sample/(µl)
1.pMWnifVSUgluDH(Extracted Plasmid)	5
2.pHYnifB~hesA,pMWnifVSUgluDH(Extracted Plasmid)	5



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1.Protocol 2.Laboratory notebook