Team:Nagahama parta

From 2014.igem.org

(Difference between revisions)
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<p><a href="http://parts.igem.org/Part:BBa_K1342004">BBa_K1342004</a></p>
<p><a href="http://parts.igem.org/Part:BBa_K1342004">BBa_K1342004</a></p>
<p><a href="http://parts.igem.org/Part:BBa_K1342005">BBa_K1342005</a></p>
<p><a href="http://parts.igem.org/Part:BBa_K1342005">BBa_K1342005</a></p>
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<br>
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<br>
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==='''Method'''===
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<br>
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'''Aspartate synthesis'''
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<br>
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 +
E.coli K12 transformed with CdP-R.B.S-AspA-d.Ter (BBa_K1342001) previous cultured with cadmium in LB medium (250μM) in 37℃ for 12hr at 120rpm. Adjust Cell mass (OD1.0) and therefor centrifuged 4000rpm for 20 min. Cell pellets ware activated in synthesis medium in 37℃ for 2hr at 120rpm/min.
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 +
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 +
TLC
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<br>
 +
・Developer
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<br>
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Ethyl acetate: pyridine: water: acetic acid＝162：21：11：6
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<br>
 +
Reagent
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 +
7mg/mL     5-(Dimethylamino) naphthalene-1-sulfonyl Chloride (in Acetone); (DNS)
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<br>
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1.Sample: DNS =1: 1
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<br>
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2.Incubate RT ＞30min
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<br>
 +
3.Spot 2μL
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<br>
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4.Development
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<br>
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5.UV irradiation (365nm)
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<br>
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'''SDS PAGE'''
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<br>
 +
・Preculture E.coli holding a plasmid containing a target gene or nomal E.coli.
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<br>
 +
・Measure OD600 0.6-1.0
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<br>
 +
・the expression of fusion protein by isopropylthio-β-D-galactoside (IPTG)　and Cd2+ soln.
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<br>
 +
・Transfer a sample a 200 µl in a microcentrifuge tube
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<br>
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・Centrifuge at 13,000rpm for a minute at 4℃
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<br>
 +
・Discard supernatant quantitative
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<br>
 +
・Store pellet at -20 °C
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<br>
 +
・Thaw pellet and resupend in Sample Buffer (100 µL 1xSample Buffer per samples)
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<br>
 +
・Heat for 5 minutes at 98 °C
 +
<br>
 +
・Centrifuge at 13,000rpm for 10 minutes at 4℃
 +
<br>
 +
・Transfer supernatant to a new microcentrifuge tube
 +
<br>
 +
・Analyze samples by SDS-PAGE.(Use 20 µL per samples)
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<br>
 +
 +
 +
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<font size=5>Chemotaxis Assay Using Capillary</font>
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<font size=3>Chemotaxis of a bacterium such as E.coli is assayed by measuring the number of organisms attracted into a capillary tube containing an attractant.</font>
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<font size=4>Protocol</font>
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<font size=3>
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Preculture JM109 using tripton broth (Incubate 50rpm/min 30℃ 12hr) Check motility under the microscope.Diluting the E.coli culture 100 times. (tripton broth:E.coli culture＝20mL:200㎕)Incubate 50rpm/min 30℃ (until log phase)500㎕ into two tubes.Centrifuge two tubes in low speed. (25℃ 10min in 3400G)Throw away the clear top of liquid.Add wash medium(500㎕)Resuspending pellets softlyDo 5~9 againAdd chemotaxis medium(500㎕)Resuspending pellets softly.Check motility under the microscope.E.coli chemotaxis medium into chamber preparation(100㎕)Prepare the positive and negative control capillary.（1㎕ of 10mM aspartate chemotaxis medium and chemotaxis medium into capillary.）Two capillary into chamber preparation.90mim 30℃ incubationDilute the chemotaxis buffer in capillary`s liquid 100 times.Prating to LBmedium.37℃ 12h incubation.Counting the colony.Finish
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</font>
</a>
</a>
</html>
</html>

Revision as of 05:43, 14 October 2014


BBa_K1342001

BBa_K1342002

BBa_K1342003

BBa_K1342004

BBa_K1342005



==='''Method'''===
'''Aspartate synthesis'''
E.coli K12 transformed with CdP-R.B.S-AspA-d.Ter (BBa_K1342001) previous cultured with cadmium in LB medium (250μM) in 37℃ for 12hr at 120rpm. Adjust Cell mass (OD1.0) and therefor centrifuged 4000rpm for 20 min. Cell pellets ware activated in synthesis medium in 37℃ for 2hr at 120rpm/min. TLC
・Developer
Ethyl acetate: pyridine: water: acetic acid＝162：21：11：6
Reagent 7mg/mL 5-(Dimethylamino) naphthalene-1-sulfonyl Chloride (in Acetone); (DNS)
1.Sample: DNS =1: 1
2.Incubate RT ＞30min
3.Spot 2μL
4.Development
5.UV irradiation (365nm)
'''SDS PAGE'''
・Preculture E.coli holding a plasmid containing a target gene or nomal E.coli.
・Measure OD600 0.6-1.0
・the expression of fusion protein by isopropylthio-β-D-galactoside (IPTG)　and Cd2+ soln.
・Transfer a sample a 200 µl in a microcentrifuge tube
・Centrifuge at 13,000rpm for a minute at 4℃
・Discard supernatant quantitative
・Store pellet at -20 °C
・Thaw pellet and resupend in Sample Buffer (100 µL 1xSample Buffer per samples)
・Heat for 5 minutes at 98 °C
・Centrifuge at 13,000rpm for 10 minutes at 4℃
・Transfer supernatant to a new microcentrifuge tube
・Analyze samples by SDS-PAGE.(Use 20 µL per samples)
Chemotaxis Assay Using Capillary Chemotaxis of a bacterium such as E.coli is assayed by measuring the number of organisms attracted into a capillary tube containing an attractant. Protocol Preculture JM109 using tripton broth (Incubate 50rpm/min 30℃ 12hr) Check motility under the microscope.Diluting the E.coli culture 100 times. (tripton broth:E.coli culture＝20mL:200㎕)Incubate 50rpm/min 30℃ (until log phase)500㎕ into two tubes.Centrifuge two tubes in low speed. (25℃ 10min in 3400G)Throw away the clear top of liquid.Add wash medium(500㎕)Resuspending pellets softlyDo 5~9 againAdd chemotaxis medium(500㎕)Resuspending pellets softly.Check motility under the microscope.E.coli chemotaxis medium into chamber preparation(100㎕)Prepare the positive and negative control capillary.（1㎕ of 10mM aspartate chemotaxis medium and chemotaxis medium into capillary.）Two capillary into chamber preparation.90mim 30℃ incubationDilute the chemotaxis buffer in capillary`s liquid 100 times.Prating to LBmedium.37℃ 12h incubation.Counting the colony.Finish

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