Team:Nagahama parta

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<a href="http://parts.igem.org/cgi/partsdb/pgroup.cgi?pgroup=iGEM2014&group=Nagahama" style="text-decoration:none;color:#1C140D">
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<a href="https://2014.igem.org/Team:Nagahama_Parts" style="text-decoration:none;color:#1C140D">
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PARTS
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Parts
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<a href="https://2014.igem.org/Team:Nagahama_H.P." style="text-decoration:none;color:#1C140D">
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<a href="https://2014.igem.org/Team:Nagahama_Policy_&_Practice" style="text-decoration:none;color:#1C140D">
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H.P.
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Policy & Practice
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<p><a href="http://parts.igem.org/Part:BBa_K1342001">BBa_K1342001</a></p>
 
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<p><a href="http://parts.igem.org/Part:BBa_K1342002">BBa_K1342002</a></p>
 
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<p><a href="http://parts.igem.org/Part:BBa_K1342003">BBa_K1342003</a></p>
 
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<p><a href="http://parts.igem.org/Part:BBa_K1342004">BBa_K1342004</a></p>
 
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<p><a href="http://parts.igem.org/Part:BBa_K1342005">BBa_K1342005</a></p>
 
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<groupparts>iGEM014 Nagahama</groupparts>
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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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TLC
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<br>
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・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>
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Reagent
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7mg/mL     5-(Dimethylamino) naphthalene-1-sulfonyl Chloride (in Acetone); (DNS)
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1.Sample: DNS =1: 1
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2.Incubate RT ＞30min
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3.Spot 2μL
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4.Development
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5.UV irradiation (365nm)
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'''SDS PAGE'''
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・Preculture E.coli holding a plasmid containing a target gene or nomal E.coli.
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<br>
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・Measure OD600 0.6-1.0
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<br>
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・the expression of fusion protein by isopropylthio-β-D-galactoside (IPTG)　and Cd2+ soln.
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<br>
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・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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・Discard supernatant quantitative
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<br>
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・Store pellet at -20 °C
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<br>
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・Thaw pellet and resupend in Sample Buffer (100 µL 1xSample Buffer per samples)
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<br>
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・Heat for 5 minutes at 98 °C
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<br>
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・Centrifuge at 13,000rpm for 10 minutes at 4℃
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・Transfer supernatant to a new microcentrifuge tube
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<br>
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・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>
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Latest revision as of 13:17, 16 October 2014


<groupparts>iGEM014 Nagahama</groupparts>

Retrieved from "http://2014.igem.org/Team:Nagahama_parta"