Difference between revisions of "Part:BBa K624032"
 
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<partinfo>BBa_K624032 short</partinfo>
 
<partinfo>BBa_K624032 short</partinfo>
  
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Facultative anaeroabic magnetotactic bacterial species, Magnetospirillum magneticum AMB-1. Grow in liquid MSGM (Magnetic Spirillum Growth Medium), or on MSGM agar plate.
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Facultative anaeroabic magnetotactic bacterial species, Magnetospirillum magneticum AMB-1. Grow in liquid MSGM (Magnetic Spirillum Growth Medium), or on MSGM agar plate.<font size=3>Like ''E. coli'', AMB-1 also grows in liquid medium, and this medium is called '''Magnetic Spirillum Growth Medium (MSGM)'''.  In the medium, we added nutrient, iron source (supplying AMB-1 in magnetite formation within magnetosomes), vitamin, and mineral solution.</font>
  
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NCBI: [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=genome&Cmd=ShowDetailView&TermToSearch=19021 whole genome sequence]
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[[Image:AMB-1.jpg]]
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'''NCBI:''' [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=genome&Cmd=ShowDetailView&TermToSearch=19021 whole genome sequence]
  
 
<!-- Add more about the biology of this part here
 
<!-- Add more about the biology of this part here
 
===Usage and Biology===
 
===Usage and Biology===
  
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=='''<font size=5><font color=crimson>Cultivation of ''Magnetospirillum magneticum'' AMB-1</font>'''</font>  <font color=gray><font size=4>  '''Actually, it was not difficult...</font></font>'''==
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<font size=3>Like ''E. coli'', AMB-1 also grows in liquid medium, and this medium is called '''Magnetic Spirillum Growth Medium (MSGM)'''.  In the medium, we added nutrient, iron source (supplying AMB-1 in magnetite formation within magnetosomes), vitamin, and mineral solution.</font>
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<font size=3>'''MSGM's  ingredients'''
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<font color=red>(In grams per liter)</font>
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●Succinic acid            0.74 g
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●KH2PO4                    0.68 g
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●Sodium nitrate            0.12 g
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●Sodium thioglycolate      0.1 g
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●[http://2011.igem.org/File:Wolfe_vitamin.pdf *Wolfe's vitamin solution]  10 ml(stock)
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●[http://2011.igem.org/File:Wolfe.pdf *Wolfe’s mineral solution]  5  ml(stock)
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●Ferric quinate solution  2  ml(0.27 g of FeCl<font size=1>3</font> and 0.19 g of quinic acid in 100 ml water)
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  '''Adjust pH to 7.0 with NaOH'''
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We also refered to an [http://www.sciencedirect.com/science/article/pii/S014102290100343X original article] published by Chen-Dong Yang'', et al.'', which added '''Polypeptone''' and '''Yeast extract''' into MSGM and added '''L-cysteine''' instead of sodium thioglycolate, a mixture called '''<font color=red>En-rich MSGM</font>'''. In <<''Effects of growth medium composition, iron sources and atmospheric oxygen concentrations on production of luciferase-bacterial magnetic particle complex by a recombinant Magnetospirillum magneticum AMB-1,2001''>> said that MSGM enriched with L-cysteine, yeast extract and polypeptone could enhance BMP (Bacterial Magnetic Particles) productivity. Addition of yeast extract had no effect on BMP (Bacterial Magnetic Particles) production and polypeptone only improved the final cell density and therefore slightly improved BMP (Bacterial Magnetic Particles) production, whereas L-cysteine induced cell growth!
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</font>
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<font size=3>'''En-rich MSGM's  ingredients'''
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<font color=red>(In grams per liter)</font>
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 +
●Succinic acid            0.74 g
 +
●KH2PO4                    0.68 g
 +
●Sodium nitrate            0.12 g
 +
●[http://2011.igem.org/File:Wolfe_vitamin.pdf *Wolfe's vitamin solution]  10 ml(stock)
 +
●[http://2011.igem.org/File:Wolfe.pdf *Wolfe’s mineral solution]  5  ml(stock)
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●Ferric quinate solution  2  ml(0.27 g of FeCl<font size=1>3</font> and 0.19 g of quinic acid in 100 ml water)
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●'''<font color=red>L-cysteine                0.005%(Instead of sodium thioglycolate) </font>'''
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●'''<font color=red>Yeast extract            0.01%</font>'''
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●'''<font color=red>Polypeptone              0.02%</font>'''
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  '''Adjust pH to 7.0 with NaOH'''
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Strict anaerobic conditions have been thought to facilitate BMP (Bacterial Magnetic Particles) production in AMB-1 cells. To induce AMB-1 forming magnetosome, we have to control low O<sub>2</sub> concentration.
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[[file:AMB1.jpg]]
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</font>
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=='''<font size=5><font color=crimson>Electroporation</font>'''</font>    <font color=gray>So...how to insert our plasmid into the AMB-1?</font>==
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<font size=3>After plasmid constructions were done, we need to transform them into AMB-1. Electroporation was performed with a '''Gene Pulser''' (Bio-Rad Laboratories, Richmond, Calif.), at a capacitance of 25 micro-F and a resistance of 200Ω, and 0.1-cm cuvettes. Electroporation allows cellular introduction of large highly charged molecules such as DNA which would never passively diffuse across the hydrophobic bilayer core. This phenomenon indicates that the mechanism is the creation of nm-scale water-filled holes in the membrane.</font>
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[[file:elem.jpg]]
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<font size=3>'''Procedure''':
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● Step1: Harvested and washed AMB-1 with 10 mM TES buffer containing 272 mM sucrose (pH 7.5).
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● Step2: Resuspended AMB-1 in the same buffer at 10^9 cells/ml.
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● Step3: Transferred to 500 micro-liter of MSGM supplemented with 20 mM Mg<font size=1>2+</font> and
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incubated at 27°C overnight with shaking at 100 rpm.
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● Step4: Diluted in 5 ml of MSGM containing 0.7% agar.
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● Step5: Plated on 1% agar in MSGM (supplied with ampicillin at 5 ug/ml or kanamycin at 2.5 ug/ml)
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incubated under anaerobic conditions.
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</font>
 
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<span class='h3bb'>Sequence and Features</span>
 
<span class='h3bb'>Sequence and Features</span>
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<partinfo>BBa_K624032 parameters</partinfo>
 
<partinfo>BBa_K624032 parameters</partinfo>
 
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We also refered to an original article published by Chen-Dong Yang, et al., which added Polypeptone and Yeast extract into MSGM and added L-cysteine instead of sodium thioglycolate, a mixture called En-rich MSGM. In <<Effects of growth medium composition, iron sources and atmospheric oxygen concentrations on production of luciferase-bacterial magnetic particle complex by a recombinant Magnetospirillum magneticum AMB-1,2001>> said that MSGM enriched with '''L-cysteine'', '''yeast extract''' and '''polypeptone''' could enhance BMP (Bacterial Magnetic Particles) productivity. Addition of yeast extract had no effect on BMP (Bacterial Magnetic Particles) production and polypeptone only improved the final cell density and therefore slightly improved BMP (Bacterial Magnetic Particles) production, whereas L-cysteine induced cell growth!
 +
 +
 +
 +
<font size=3>'''MSGM's  ingredients'''
 +
 +
 +
<font color=red>(In grams per liter)</font>
 +
 +
●Succinic acid            0.74 g
 +
●KH2PO4                    0.68 g
 +
●Sodium nitrate            0.12 g
 +
●Sodium thioglycolate      0.1 g
 +
●[http://2011.igem.org/File:Wolfe_vitamin.pdf *Wolfe's vitamin solution]  10 ml(stock)
 +
●[http://2011.igem.org/File:Wolfe.pdf *Wolfe’s mineral solution]  5  ml(stock)
 +
●Ferric quinate solution  2  ml(0.27 g of FeCl<font size=1>3</font> and 0.19 g of quinic acid in 100 ml water)
 +
  '''Adjust pH to 7.0 with NaOH'''
 +
 +
 +
 +
'''En-rich MSGM's ingredients'''
 +
 +
 +
<font color=red>(In grams per liter)</font>
 +
 +
●Succinic acid            0.74 g
 +
●KH2PO4                    0.68 g
 +
●Sodium nitrate            0.12 g
 +
●*Wolfe's vitamin solution  10 ml(stock)
 +
●*Wolfe’s mineral solution  5  ml(stock)
 +
●Ferric quinate solution  2  ml(0.27 g of FeCl3 and 0.19 g of quinic acid in 100 ml water)
 +
●L-cysteine                0.005%(Instead of sodium thioglycolate)
 +
●Yeast extract            0.01%
 +
●Polypeptone              0.02%
 +
Adjust pH to 7.0 with NaOH
 +
 +
Strict anaerobic conditions have been thought to facilitate BMP (Bacterial Magnetic Particles) production in AMB-1 cells. To induce AMB-1 forming magnetosome, we have to control low O2 concentration.
 +
 +
 +
 +
 +
'''Growth Curve Of AMB-1'''
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We measure AMB-1's growth under '''OD565''', which is the best absorption wavelength for AMB-1. For electroporation operation, it is important to find out the log phase, since it’s the best time for electroporation. By keeping AMB-1 in "log phase" (approximately '''3~6 days in Enrich-MSGM''', '''7~10 days in MSGM'''), best results of electroporation are achieved. And after seven days, the bacterium would go into stationary phase. During the phase, the growth slows as a result of nutrient depletion and accumulation of toxic products. Growth rate and death rate of bacterium is best likely to meet equilibrium in this stage.
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[[Image:AMB-1curve.jpg]]

Latest revision as of 07:28, 9 October 2011

Magnetospirillum magneticum AMB-1

Facultative anaeroabic magnetotactic bacterial species, Magnetospirillum magneticum AMB-1. Grow in liquid MSGM (Magnetic Spirillum Growth Medium), or on MSGM agar plate.Like E. coli, AMB-1 also grows in liquid medium, and this medium is called Magnetic Spirillum Growth Medium (MSGM). In the medium, we added nutrient, iron source (supplying AMB-1 in magnetite formation within magnetosomes), vitamin, and mineral solution.

AMB-1.jpg




NCBI: [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=genome&Cmd=ShowDetailView&TermToSearch=19021 whole genome sequence]

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]


We also refered to an original article published by Chen-Dong Yang, et al., which added Polypeptone and Yeast extract into MSGM and added L-cysteine instead of sodium thioglycolate, a mixture called En-rich MSGM. In <<Effects of growth medium composition, iron sources and atmospheric oxygen concentrations on production of luciferase-bacterial magnetic particle complex by a recombinant Magnetospirillum magneticum AMB-1,2001>> said that MSGM enriched with L-cysteine, yeast extract' and polypeptone could enhance BMP (Bacterial Magnetic Particles) productivity. Addition of yeast extract had no effect on BMP (Bacterial Magnetic Particles) production and polypeptone only improved the final cell density and therefore slightly improved BMP (Bacterial Magnetic Particles) production, whereas L-cysteine induced cell growth!


MSGM's ingredients


(In grams per liter) 

●Succinic acid             0.74 g
●KH2PO4                    0.68 g
●Sodium nitrate            0.12 g
●Sodium thioglycolate      0.1 g
●[http://2011.igem.org/File:Wolfe_vitamin.pdf *Wolfe's vitamin solution]  10 ml(stock)
●[http://2011.igem.org/File:Wolfe.pdf *Wolfe’s mineral solution]  5  ml(stock)
●Ferric quinate solution   2  ml(0.27 g of FeCl3 and 0.19 g of quinic acid in 100 ml water)
 Adjust pH to 7.0 with NaOH


En-rich MSGM's ingredients


(In grams per liter) 

●Succinic acid             0.74 g
●KH2PO4                    0.68 g
●Sodium nitrate            0.12 g
●*Wolfe's vitamin solution  10 ml(stock)
●*Wolfe’s mineral solution  5  ml(stock)
●Ferric quinate solution   2  ml(0.27 g of FeCl3 and 0.19 g of quinic acid in 100 ml water)
●L-cysteine                0.005%(Instead of sodium thioglycolate) 
●Yeast extract             0.01%
●Polypeptone               0.02%
Adjust pH to 7.0 with NaOH

Strict anaerobic conditions have been thought to facilitate BMP (Bacterial Magnetic Particles) production in AMB-1 cells. To induce AMB-1 forming magnetosome, we have to control low O2 concentration.



Growth Curve Of AMB-1

We measure AMB-1's growth under OD565, which is the best absorption wavelength for AMB-1. For electroporation operation, it is important to find out the log phase, since it’s the best time for electroporation. By keeping AMB-1 in "log phase" (approximately 3~6 days in Enrich-MSGM, 7~10 days in MSGM), best results of electroporation are achieved. And after seven days, the bacterium would go into stationary phase. During the phase, the growth slows as a result of nutrient depletion and accumulation of toxic products. Growth rate and death rate of bacterium is best likely to meet equilibrium in this stage.

AMB-1curve.jpg