[[File:Streptococcus equinus B315 scanning electron micrograph by Ahmed Hetta.jpg|thumb|350x350px|Streptococcus equinus B315 scanning electron micrograph by Ahmed Hetta]]

== Introduction ==
'''''Streptococcus equinus'' B315'''

''Streptococcus equinus'' B315 was isolated from bovine rumen contents in NZ [1]. B315 is proteolytic, Gram positive, and able to ferment a wide range of carbohydrates. Its main fermentation product is lactate, together with succinate and acetate [1]. The B315 genome encodes two lantibiotic bacteriocin biosynthetic gene clusters.

The draft genome for B315 (1,786,673 bp) was generated through the Hungate1000 project with JGI [2].

NCBI: ''Streptococcus equinus''.

GTDB: ''Streptococcus equinus''. B315 belongs to the same species group as the ''S. equinus'' type strain (DSM 20558).

Risk group: 2.

== Global Methane Hub Metadata ==
{| class="wikitable"
|+
|Origin:
|New Zealand
|-
|Sequenced by:
|Ahmed Hetta and Yuqi Li
|-
|Assembled by:
|William Alexander
|-
|Methylome called by:
|William Alexander
|-
|Methyltransferases cloned by:
|
|-
|MIC and Culturing Conditions by:
|
|-
|Transformation by:
|Ahmed Hetta
|}

== Genome ==

== DefenseFinder Results ==

== Methylome ==
{| class="wikitable"
| valign="top" |'''#6mA modified motifs'''
| valign="top" |
| valign="top" |
| valign="top" |
|-
| valign="top" |'''#motif'''
| valign="top" |methylCounts
| valign="top" |totalCounts
| valign="top" |%motifsMethyl
|-
| valign="top" |'''GAR(6mA)NNNNNNNTAC'''
| valign="top" |412
| valign="top" |430
| valign="top" |95.81
|-
| valign="top" |'''GT(6mA)NNNNNNNTYTC'''
| valign="top" |413
| valign="top" |430
| valign="top" |96.05
|}

== Methylating E. coli Strains ==

== Culturing Conditions ==

== Antibiotic MIC Tests ==

== Protocols ==

== Transformation Results ==

== External Links ==
[1] Attwood GT, Reilly K. 1995. Identification of proteolytic rumen bacteria isolated from New Zealand cattle. J Appl Bacteriol 79: 22-9. doi: 10.1111/j.1365-2672.1995.tb03119.x.

[1] Seshadri R, Leahy SC, Attwood GT, Teh KH, Lambie SC, Cookson AL, Eloe-Fadrosh EA, Pavlopoulos GA, Hadjithomas M, Varghese NJ, Paez-Espino D; Hungate1000 project collaborators; Perry R, Henderson G, Creevey CJ, Terrapon N, Lapebie P, Drula E, Lombard V, Rubin E, Kyrpides NC, Henrissat B, Woyke T, Ivanova NN, Kelly WJ. 2019. Cultivation and sequencing of rumen microbiome members from the Hungate1000 Collection. Nature Biotechnol 36: 359-367. doi: 10.1038/nbt.4110.

Streptococcus equinus B315

2026-10-29T16:56:06Z

Ahetta:

File:Streptococcus equinus B315 scanning electron micrograph by Ahmed Hetta.jpg

2026-10-29T16:54:52Z

Ahetta:

Streptococcus equinus B315 scanning electron micrograph by Ahmed Hetta

Streptococcus equinus B315

2026-10-29T16:50:58Z

Ahetta: /* Global Methane Hub Metadata */

== Introduction ==
'''''Streptococcus equinus'' B315'''

''Streptococcus equinus'' B315 was isolated from bovine rumen contents in NZ [1]. B315 is proteolytic, Gram positive, and able to ferment a wide range of carbohydrates. Its main fermentation product is lactate, together with succinate and acetate [1]. The B315 genome encodes two lantibiotic bacteriocin biosynthetic gene clusters.

The draft genome for B315 (1,786,673 bp) was generated through the Hungate1000 project with JGI [2].

NCBI: ''Streptococcus equinus''.

GTDB: ''Streptococcus equinus''. B315 belongs to the same species group as the ''S. equinus'' type strain (DSM 20558).

Risk group: 2.

== Global Methane Hub Metadata ==
{| class="wikitable"
|+
|Origin:
|New Zealand
|-
|Sequenced by:
|Ahmed Hetta and Yuqi Li
|-
|Assembled by:
|William Alexander
|-
|Methylome called by:
|William Alexander
|-
|Methyltransferases cloned by:
|
|-
|MIC and Culturing Conditions by:
|
|-
|Transformation by:
|Ahmed Hetta
|}

== Genome ==

== DefenseFinder Results ==

== Methylome ==

== Methylating E. coli Strains ==

== Culturing Conditions ==

== Antibiotic MIC Tests ==

== Protocols ==

== Transformation Results ==

== External Links ==
[1] Attwood GT, Reilly K. 1995. Identification of proteolytic rumen bacteria isolated from New Zealand cattle. J Appl Bacteriol 79: 22-9. doi: 10.1111/j.1365-2672.1995.tb03119.x.

[1] Seshadri R, Leahy SC, Attwood GT, Teh KH, Lambie SC, Cookson AL, Eloe-Fadrosh EA, Pavlopoulos GA, Hadjithomas M, Varghese NJ, Paez-Espino D; Hungate1000 project collaborators; Perry R, Henderson G, Creevey CJ, Terrapon N, Lapebie P, Drula E, Lombard V, Rubin E, Kyrpides NC, Henrissat B, Woyke T, Ivanova NN, Kelly WJ. 2019. Cultivation and sequencing of rumen microbiome members from the Hungate1000 Collection. Nature Biotechnol 36: 359-367. doi: 10.1038/nbt.4110.

Streptococcus bovis JB1

2026-10-29T16:48:25Z

Ahetta:

[[File:S. bovis SEM.jpg|thumb|350x350px|Scanning electron micrograph of ''Streptococcus bovis'' JB1 by Ahmed Hetta]]

== Introduction ==
''Streptococcus bovis'' JB1 was isolated from a cow fed alfalfa hay in Davis, CA, USA1. It is a major amylolytic bacterium that depolymerize starch to glucose, fermenting it to large amounts of lactic acid that can lead to lactic acidosis in ruminants.

== Global Methane Hub Metadata ==
{| class="wikitable"
|+
|Origin:
|Illinois, Mackie Lab
|-
|Sequenced by:
|Ahmed Hetta and Yuqi Li
|-
|Assembled by:
|William G. Alexander
|-
|Methylome called by:
|William G. Alexander
|-
|Methyltransferases cloned by:
|No methylation motifs have been detected
|-
|MIC and Culturing Conditions by:
|Ahmed Hetta and Yuqi Li
|-
|Transformation by:
|Ahmed Hetta
|}

== Genome ==
[[File:Streptococcus_bovis_JB1_1_1_circos.png|center|thumb|Circos plot of ''Streptococcus bovis'' JB1|400x400px]]

== DefenseFinder Results ==
== Methylome ==
No methylation motifs were detected in ''Streptococcus bovis'' JB1.

== Methylating E. coli Strains ==
No ''E. coli'' methylation strain has been constructed for ''S. bovis'' JB1 strain

== Culturing Conditions ==
''Streptococcus bovis'' JB1 grows on both M2GSC and [[SDM culture medium|SDM]] (Semi Defined medium). Cells grow overnight at 37 °C in SDM or M2GSC culture medium with head space composed of 95% CO2 and 5% H2. Similarly, ''Streptococcus bovis'' JB1 grows overnight on SDM agar plates. Agar plates were placed in anaerobic Jar and incubated in anaerobic chamber under gas mixture that is composed of 95% CO2 and 5% H2.

== Antibiotic MIC Tests ==
[[File:Streptococcus_bovis_JB1_MIC.png|left|thumb|MIC tests of ''Streptococcus bovis'' JB1|375x375px]]
{| class="wikitable"
!antibiotic
!concentration (ug/mL)
|-
|Ampicillin
|0.25
|-
|Kanamycin
|Resistant (>200 ug/mL)
|-
|Chloramphenicol
|0.50
|-
|Tetracycline
|0.23
|-
|Gentamicin
|1.0
|-
|Streptomycin
|Resistant (>200 ug/mL)
|-
|Erythromycin
|1
|}

== Protocols ==

* '''[[SDM culture medium|Preparation of SDM culture medium]]'''
* '''Preparation of M2GSC culture medium'''

* [[Streptococcus transformation protocol|'''Transformation protocol''']]

== Transformation Results ==

== External Links ==

# arvis GN, Kurtovic A, Hay AG, Russell JB. The physiological and genetic diversity of bovine Streptococcus bovis strains(1). FEMS Microbiol Ecol. 2001 Mar;35(1):49-56. doi: 10.1016/s0168-6496(00)00110-0. PMID: 11248389.

File:S. bovis SEM.jpg

2026-10-29T16:45:19Z

Ahetta:

Scanning electron micrograph of Streptococcus bovis JB1

Streptococcus bovis JB1

2026-10-29T01:15:11Z

Ahetta:

Streptococcus bovis JB1

2026-10-27T23:20:33Z

Ahetta:

Streptococcus bovis JB1

2026-10-27T23:19:22Z

Ahetta:

Streptococcus bovis JB1

2026-10-27T23:15:38Z

Ahetta:

[[File:SEM_electron_micrograph_of_Streptococcus_bovis_JB1.jpg|thumb|300x300px|''S. azabuensis'' KH1P5 in liquid culture exhibiting its dense layer of EPS. Source: Dr. Roy Rodriguez Carrero]]

[[File:S. bovis 1.jpg|alt=kk|thumb|kkk]]

== Introduction ==
''Streptococcus bovis'' JB1 was isolated from the rumen of alfalfa-fed cow. It is a major amylolytic bacterium that depolymerize starch to glucose, fermenting it to large amounts of lactic acid that can lead to lactic acidosis in ruminants.

== Global Methane Hub Metadata ==
{| class="wikitable"
|+
|Origin:
|Illinois, Mackie Lab
|-
|Sequenced by:
|Ahmed Hetta and Yuqi Li
|-
|Assembled by:
|William G. Alexander
|-
|Methylome called by:
|William G. Alexander
|-
|Methyltransferases cloned by:
|No methylation motifs have been detected
|-
|MIC and Culturing Conditions by:
|Ahmed Hetta and Yuqi Li
|-
|Transformation by:
|Ahmed Hetta
|}

== Genome ==
[[File:Streptococcus_bovis_JB1_1_1_circos.png|center|thumb|Circos plot of ''S. azabuensis'' KH1P5. Source: Dr. William G. Alexander]]

== DefenseFinder Results ==
== Methylome ==
No methylation motifs were detected in ''Streptococcus bovis'' JB1.

== Methylating E. coli Strains ==
No ''E. coli'' methylation strain has been constructed for ''S. bovis'' JB1 strain

== Culturing Conditions ==
''Streptococcus bovis'' JB1 grows on both M2GSC and [[SDM culture medium|SDM]] (Semi Defined medium). Cells grow overnight at 37 °C in SDM or M2GSC culture medium with head space composed of 95% CO2 and 5% H2. Similarly, ''Streptococcus bovis'' JB1 grows overnight on SDM agar plates. Agar plates were placed in anaerobic Jar and incubated in anaerobic chamber under gas mixture that is composed of 95% CO2 and 5% H2.

== Antibiotic MIC Tests ==
[[File:Streptococcus_bovis_JB1_MIC.png|left|thumb|MIC tests of ''Streptococcus bovis'' JB1|375x375px]]
{| class="wikitable"
!antibiotic
!concentration (ug/mL)
|-
|Ampicillin
|0.25
|-
|Kanamycin
|Resistant (>200 ug/mL)
|-
|Chloramphenicol
|0.50
|-
|Tetracycline
|0.23
|-
|Gentamicin
|1.0
|-
|Streptomycin
|Resistant (>200 ug/mL)
|-
|Erythromycin
|1
|}

== Protocols ==

* '''[[SDM culture medium|Preparation of SDM culture medium]]'''
* '''Preparation of M2GSC culture medium'''

* [[Streptococcus transformation protocol|'''Transformation protocol''']]

== Transformation Results ==

== External Links ==
[1] Seshadri R, Leahy SC, Attwood GT, Teh KH, Lambie SC, Cookson AL, Eloe-Fadrosh EA, Pavlopoulos GA, Hadjithomas M, Varghese NJ, Paez-Espino D; Hungate1000 project collaborators; Perry R, Henderson G, Creevey CJ, Terrapon N, Lapebie P, Drula E, Lombard V, Rubin E, Kyrpides NC, Henrissat B, Woyke T, Ivanova NN, Kelly WJ. 2019. Cultivation and sequencing of rumen microbiome members from the Hungate1000 Collection. Nature Biotechnol 36: 359-367. doi: 10.1038/nbt.4110.

[2] Kumar S, Treloar BP, Teh KH, McKenzie CM, Henderson G, Attwood GT, Waters SM, Patchett ML, Janssen PH. 2018. Sharpea and Kandleria are lactic acid producing rumen bacteria that do not change their fermentation products when co-cultured with a methanogen. Anaerobe 54: 31-38. doi: 10.1016/j.anaerobe.2018.07.008.

[3] Kittelmann S, Pinares-Patiño CS, Seedorf H, Kirk MR, Ganesh S, McEwan JC, Janssen PH. 2014. Two different bacterial community types are linked with the low-methane emission trait in sheep. PLoS One 9: e103171. doi: 10.1371/journal.pone.0103171.

[4] Kamke J, Kittelmann S, Soni P, Li Y, Tavendale M, Ganesh S, Janssen PH, Shi W, Froula J, Rubin EM, Attwood GT. 2016. Rumen metagenome and metatranscriptome analyses of low methane yield sheep reveals a Sharpea-enriched microbiome characterised by lactic acid formation and utilisation. Microbiome 4: 56. doi: 10.1186/s40168-016-0201-2.

[5] Morita H, Shiratori C, Murakami M, Takami H, Toh H, Kato Y, Nakajima F, Takagi M, Akita H, Masaoka T, Hattori M. 2008. Sharpea azabuensis gen. nov., sp. nov., a Gram-positive, strictly anaerobic bacterium isolated from the faeces of thoroughbred horses. Int J Syst Evol Microbiol 58: 2682-2686 doi: 10.1099/ijs.0.65543-0.

Streptococcus bovis JB1

2026-10-27T23:14:42Z

Ahetta: /* Culturing Conditions */

File:S. bovis 1.jpg

2026-10-27T23:13:36Z

Ahetta:

Streptococcus bovis JB1 scanning electron micrograph

Streptococcus transformation protocol

2026-10-27T23:10:32Z

Ahetta: Streptococcus transformation protocol

'''Culture media:'''

'''1. For overnight culture (SGM17):'''

Beef extract ……………….. 5.00 g/l

Polypeptone ……………… 5.00 g/l

Phytone-peptone …………… 5.00g/l

Yeast extract ……………….. 2.50 g/l

Disodium glycerol phosphate 19.00 g/l

Ascorbic acid ………………...0.50 g/l

Glucose ………………………5.00 g/l

MgSO4.7H2O ………………. 0.25 g/l

+

0.5 M Sucrose (171.15 g/l)

- Filter-sterilize

'''2. For growing the cells to make electrocompetent cells:'''

SGM17 (above) + '''0.5% glycine'''

'''3. For recovery after electroporation:'''

SGM17 containing '''20 mM MgCl2''' and '''2 mM CaCl2'''

'''4. For plating:'''

'''SR medium contains (g/l):''' (Supplement with erythromycin (5 and 10 mg/ml)

- Tryptone 10

- Yeast extract 5.0

- Sucrose 200

- glucose 10

- Gelatin, 25

- agar 15

- MgCl 2.5 mM

- CaCl2 2.5 mM

<nowiki>*</nowiki>Pour plates in the anaerobic chamber.

'''Method:'''

'''Day1'''. Grow Streptococcus in 10 ml of GM17+Sucrose culture medium overnight aerobically.

'''Day2'''. '''Preparation of competent cells'''

- Inoculate prewarmed 250 ml of GM17+Sucrose +'''0.5% glycine''' culture medium with 5 ml of the overnight culture.

- Grow aerobically at 37 °C to an OD600 of 0.2 (approximately 5 to 7 h).

- Place the culture on ice for 30 minutes and centrifuge at 5000 g, 4 °C, for 15 min.

- Wash the cell pellets three times in 25 ml of ice-cold 0.5 M sucrose/10% glycerol, and centrifuge as before''.''

- Resuspend the cell pellet in 0.5 ml of 0.5 M sucrose/10% glycerol and either use immediately for electroporation or store at -80 °C.

'''Electroporation:'''

- Mix 100 ml of the cell suspension was mixed with up to 5 ml of DNA and place in ice-cold 0.2-cm electroporation cuvette (Bio-Rad Laboratories, Richmond, CA).

- Electroporate at 2.5 kV, 200 W, 25 mF using the Bio-Rad Gene Pulser.

- Immediately after electroporation, add 0.9 ml of SGM17 containing '''20 mM MgCl2 and 2 mM CaCl2''' to the cell suspension.

- Transfer to a tube and incubate the mixture at 37 °C for 1 h.

- Dilutions of the cells were plated on SR and SDM agar plates supplemented with antibiotic (5 mg/ml erythromycin).

- Incubate in the anaerobic chamber and incubate at 37 °C for 1-2 days.

Streptococcus bovis JB1

2026-10-27T22:52:37Z

Ahetta: /* Protocols */

[[File:SEM_electron_micrograph_of_Streptococcus_bovis_JB1.jpg|thumb|300x300px|''S. azabuensis'' KH1P5 in liquid culture exhibiting its dense layer of EPS. Source: Dr. Roy Rodriguez Carrero]]

== Introduction ==
''Streptococcus bovis'' JB1 was isolated from the rumen of alfalfa-fed cow. It is a major amylolytic bacterium that depolymerize starch to glucose, fermenting it to large amounts of lactic acid that can lead to lactic acidosis in ruminants.

== Global Methane Hub Metadata ==
{| class="wikitable"
|+
|Origin:
|Illinois, Mackie Lab
|-
|Sequenced by:
|Ahmed Hetta and Yuqi Li
|-
|Assembled by:
|William G. Alexander
|-
|Methylome called by:
|William G. Alexander
|-
|Methyltransferases cloned by:
|No methylation motifs have been detected
|-
|MIC and Culturing Conditions by:
|Ahmed Hetta and Yuqi Li
|-
|Transformation by:
|Ahmed Hetta
|}

== Genome ==
[[File:Streptococcus_bovis_JB1_1_1_circos.png|center|thumb|Circos plot of ''S. azabuensis'' KH1P5. Source: Dr. William G. Alexander]]

== DefenseFinder Results ==
== Methylome ==
No methylation motifs were detected in ''Streptococcus bovis'' JB1.

== Methylating E. coli Strains ==
No ''E. coli'' methylation strain has been constructed for ''S. bovis'' JB1 strain

== Culturing Conditions ==
''Streptococcus bovis'' JB1 grows on both M2GSC and SDM (Semi Defined medium). Cells grow overnight at 37 °C in SDM or M2GSC culture medium with head space composed of 95% CO2 and 5% H2. Similarly, ''Streptococcus bovis'' JB1 grows overnight on SDM agar plates. Agar plates were placed in anaerobic Jar and incubated in anaerobic chamber under gas mixture that is composed of 95% CO2 and 5% H2.

== Antibiotic MIC Tests ==
[[File:Streptococcus_bovis_JB1_MIC.png|left|thumb|MIC tests of ''Streptococcus bovis'' JB1|375x375px]]
{| class="wikitable"
!antibiotic
!concentration (ug/mL)
|-
|Ampicillin
|0.25
|-
|Kanamycin
|Resistant (>200 ug/mL)
|-
|Chloramphenicol
|0.50
|-
|Tetracycline
|0.23
|-
|Gentamicin
|1.0
|-
|Streptomycin
|Resistant (>200 ug/mL)
|-
|Erythromycin
|1
|}

== Protocols ==
'''Preparation of [[SDM culture medium|SDM]] culture medium:'''

* '''Transformation protocol'''

'''Culture media:'''

'''1. For overnight culture (SGM17):'''

Beef extract ……………….. 5.00 g/l

Polypeptone ……………… 5.00 g/l

Phytone-peptone …………… 5.00g/l

Yeast extract ……………….. 2.50 g/l

Disodium glycerol phosphate 19.00 g/l

Ascorbic acid ………………...0.50 g/l

Glucose ………………………5.00 g/l

MgSO4.7H2O ………………. 0.25 g/l

+

0.5 M Sucrose (171.15 g/l)

- Filter-sterilize

'''2. For growing the cells to make electrocompetent cells:'''

SGM17 (above) + '''0.5% glycine'''

'''3. For recovery after electroporation:'''

SGM17 containing '''20 mM MgCl2''' and '''2 mM CaCl2'''

'''4. For plating:'''

SDM culture medium + 1.5% agar

'''Method:'''

'''Day1'''. Grow ''Streptococcus'' ''bovis'' JB1 in 10 ml of GM17+Sucrose culture medium overnight aerobically.

'''Day2'''. '''Preparation of competent cells'''

- Inoculate prewarmed 250 ml of GM17+Sucrose +'''0.5% glycine''' culture medium with 5 ml of the overnight culture.

- Grow aerobically at 37 °C to an OD600 of 0.2 (approximately 5 to 7 h).

- Place the culture on ice for 30 minutes and centrifuge at 5000 g, 4 °C, for 15 min.

- Wash the cell pellets three times in 25 ml of ice-cold 0.5 M sucrose/10% glycerol, and centrifuge as before''.''

- Resuspend the cell pellet in 0.5 ml of 0.5 M sucrose/10% glycerol and either use immediately for electroporation or store at -80 °C.

'''Electroporation:'''

- Mix 100 ml of the cell suspension was mixed with up to 5 ml of DNA and place in ice-cold 0.2-cm electroporation cuvette (Bio-Rad Laboratories, Richmond, CA).

- Electroporate at 2.5 kV, 200 W, 25 mF using the Bio-Rad Gene Pulser.

- Immediately after electroporation, add 0.9 ml of SGM17 containing '''20 mM MgCl2 and 2 mM CaCl2''' to the cell suspension.

- Transfer to a tube and incubate the mixture at 37 °C for 1 h.

- Dilutions of the cells were plated on SR and SDM agar plates supplemented with antibiotic (5 mg/ml erythromycin).

- Incubate in the anaerobic chamber and incubate at 37 °C for 1-2 days.

== Transformation Results ==

== External Links ==
[1] Seshadri R, Leahy SC, Attwood GT, Teh KH, Lambie SC, Cookson AL, Eloe-Fadrosh EA, Pavlopoulos GA, Hadjithomas M, Varghese NJ, Paez-Espino D; Hungate1000 project collaborators; Perry R, Henderson G, Creevey CJ, Terrapon N, Lapebie P, Drula E, Lombard V, Rubin E, Kyrpides NC, Henrissat B, Woyke T, Ivanova NN, Kelly WJ. 2019. Cultivation and sequencing of rumen microbiome members from the Hungate1000 Collection. Nature Biotechnol 36: 359-367. doi: 10.1038/nbt.4110.

[2] Kumar S, Treloar BP, Teh KH, McKenzie CM, Henderson G, Attwood GT, Waters SM, Patchett ML, Janssen PH. 2018. Sharpea and Kandleria are lactic acid producing rumen bacteria that do not change their fermentation products when co-cultured with a methanogen. Anaerobe 54: 31-38. doi: 10.1016/j.anaerobe.2018.07.008.

[3] Kittelmann S, Pinares-Patiño CS, Seedorf H, Kirk MR, Ganesh S, McEwan JC, Janssen PH. 2014. Two different bacterial community types are linked with the low-methane emission trait in sheep. PLoS One 9: e103171. doi: 10.1371/journal.pone.0103171.

[4] Kamke J, Kittelmann S, Soni P, Li Y, Tavendale M, Ganesh S, Janssen PH, Shi W, Froula J, Rubin EM, Attwood GT. 2016. Rumen metagenome and metatranscriptome analyses of low methane yield sheep reveals a Sharpea-enriched microbiome characterised by lactic acid formation and utilisation. Microbiome 4: 56. doi: 10.1186/s40168-016-0201-2.

[5] Morita H, Shiratori C, Murakami M, Takami H, Toh H, Kato Y, Nakajima F, Takagi M, Akita H, Masaoka T, Hattori M. 2008. Sharpea azabuensis gen. nov., sp. nov., a Gram-positive, strictly anaerobic bacterium isolated from the faeces of thoroughbred horses. Int J Syst Evol Microbiol 58: 2682-2686 doi: 10.1099/ijs.0.65543-0.

SDM culture medium

2026-10-27T22:51:01Z

Ahetta: Created page with "* '''Preparation of SDM culture medium:''' '''Mineral Sol. 1 (1L)''' {| class="wikitable" |K2HPO4 |6 g |} '''Mineral Sol. 2 (1L)''' {| class="wikitable" |NaCl |12 g |- |KH2PO4 |6 g |- |CaCl2・2H2O |1.2 g |- |MgSO4・7H2O |1.2 g |} '''VFA Sol. (per liter)''' {| class="wikitable" |Acetic acid |13.7 ml |- |Propionic acid |6 ml |- |Butyric acid |3.68 ml |- |Isobut..."

* '''Preparation of [[SDM culture medium|SDM]] culture medium:'''

'''Mineral Sol. 1 (1L)'''
{| class="wikitable"
|K2HPO4
|6 g
|}
'''Mineral Sol. 2 (1L)'''
{| class="wikitable"
|NaCl
|12 g
|-
|KH2PO4
|6 g
|-
|CaCl2・2H2O
|1.2 g
|-
|MgSO4・7H2O
|1.2 g
|}
'''VFA Sol. (per liter)'''
{| class="wikitable"
|Acetic acid
|13.7 ml
|-
|Propionic acid
|6 ml
|-
|Butyric acid
|3.68 ml
|-
|Isobutyric acid
|1.1 ml
|-
|n-valeric acid
|1.1 ml
|-
|isovaleric acids
|1.1 ml
|-
|2-methylbutyric acid
|0.94 ml
|-
|Phenylacetic acid (25uM)
|3.4 mg
|-
|Phenylpropionic acid (25uM)
|3.75 mg
|-
|0.2N NaOH
|700 ml
|}
<nowiki>*</nowiki>Adjust pH7.5 and adjust volume to 1L

'''Cysteine Sulfide Sol. (200ml)'''
{| class="wikitable"
|NaOH
|1.25 g
|-
|Cysteine-HCl
|5 g
|-
|Na2S.9H2O
|5 g
|-
|ddH2O
|200 ml
|}
- Dissolve NaOH in H2O, then boil under N2,

- Cool to RT and add Cysteine-HCl and Na2S.9H2O

'''Note:''' Cystine-HCl (0.5 g/L) and sodium sulfide (0.5 g/L) can be added directly to the medium.

'''Hemin Sol. (100ml)'''
{| class="wikitable"
|Hemin
|50 mg
|-
|1N NaOH
|1 ml
|}
- Make to 100 ml with ddH2O

- Autoclave

'''Final mixture (1L)'''
{| class="wikitable"
|Mineral Sol. 1
|50 ml
|-
|Mineral Sol. 2
|50 ml
|-
|Resazurin (0.1%)
|1 ml
|-
|Glucose
|4 g
|-
|(NH4)2SO4 for N source
|4 g
|-
|Hemin Sol. (0.1%)
|0.5 ml
|-
|Trypticase
|2 g
|-
|Yeast extract
|0.5 g
|}
Boiling and cool down with CO2 gas

'''Then add inside the anaerobic chamber'''
{| class="wikitable"
|VFA sol.
|10 ml
|-
|NaHCO3
|4 g
|-
|Cysteine sulfide Sol.
|20 ml
|}
- Adjust pH 6.7 to 6.8 inside the chamber

- Move bottle into anaerobic chamber, aliquoting medium

- Autoclave

2026-10-27T17:41:45Z

Ahetta: Created page with " == Introduction == add here == Global Methane Hub Metadata == add here == Genome == add here == DefenseFinder Results == add here == Methylome == add here == Methylating ''E. coli'' == add here == Culturing Conditions == add here == Antimicrobial MIC Tests == add here == Protocols == add here == Transformation Results == add here == External Links == add here"

== Introduction ==
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== Global Methane Hub Metadata ==
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== Genome ==
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== DefenseFinder Results ==
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== Methylome ==
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== Methylating ''E. coli'' ==
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== Culturing Conditions ==
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== Antimicrobial MIC Tests ==
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== Protocols ==
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== Transformation Results ==
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== External Links ==
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