Team:Nagahama parta

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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 <br>
+<TABLE width='925' border='0' cellspacing='0' cellpadding='0' style='border: none;'><TR><TD nowrap width='30%' style='padding-right: 2em;text-align: left;'>Samples:<TD align='center'><TD width='30%' align='right'></TABLE>
+<TABLE id='table_contents' width='915' border='0' cellspacing='0' cellpadding='0' style='position:relative;margin-left: 10px;border: 1px solid gray;background-color: #fda; padding-left: 0px;padding-right:2px;padding-top:3px;line-height: 150%; ' >
+<style type='text/css'>
+    th {line-height:100%;font-size:100%;
+        padding:5px 0; text-align:left; }
+</style>
+<TR><TH style='width:35px;text-align:center;padding-right:10px;'>Tube<TH style='width:100px;'>Part<TH style='width:80px;'>Plasmid Backbone<TH style='width:60px;'>Resistance&nbsp;<TH style='width:90px;'>Status<TH style='width:auto;'>Notes<TH style='width:140px;'>&nbsp;</TABLE><DIV style='height: 3px'></DIV>
+<TABLE id='table_contents' width='915' border='0' cellspacing='0' cellpadding='0' style='position:relative;margin-left: 10px;border: 1px solid gray;background-color: #fda; padding-left: 0px;padding-right:2px;padding-top:3px;line-height: 150%; ' >
+<TABLE_BREAK>
+<!-- Subtable for 80800 -->
+<TR><TD style='width:35px;text-align:center;padding-right:10px;'>1<TD style='width:100px;'><A class='noul_link part_link' href='http://parts.igem.org/wiki/index.php?title=Part:BBa_K1342001'>BBa_K1342001</A><TD style='width:80px;'><A class='noul_link' href='http://parts.igem.org/wiki/index.php?title=Part:pSB1C3'>pSB1C3</A><TD style='width:60px;'><TD style='width:90px;'>Accepted<TD style='width:auto;'>Sending Plasmid Backbone<BR><TD style='width:140px;'>&nbsp;</TABLE><DIV style='height: 3px'></DIV>
+<TABLE id='table_contents' width='915' border='0' cellspacing='0' cellpadding='0' style='position:relative;margin-left: 10px;border: 1px solid gray;background-color: #fda; padding-left: 0px;padding-right:2px;padding-top:3px;line-height: 150%; ' >
+<TABLE_BREAK>
+<!-- Subtable for 80801 -->
+<TR><TD style='width:35px;text-align:center;padding-right:10px;'>2<TD style='width:100px;'><A class='noul_link part_link' href='http://parts.igem.org/wiki/index.php?title=Part:BBa_K1342002'>BBa_K1342002</A><TD style='width:80px;'><A class='noul_link' href='http://parts.igem.org/wiki/index.php?title=Part:pSB1C3'>pSB1C3</A><TD style='width:60px;'><TD style='width:90px;'>Accepted<TD style='width:auto;'>Sending Plasmid Backbone<BR><TD style='width:140px;'>&nbsp;</TABLE><DIV style='height: 3px'></DIV>
+<TABLE id='table_contents' width='915' border='0' cellspacing='0' cellpadding='0' style='position:relative;margin-left: 10px;border: 1px solid gray;background-color: #fda; padding-left: 0px;padding-right:2px;padding-top:3px;line-height: 150%; ' >
+<TABLE_BREAK>
+<!-- Subtable for 80802 -->
+<TR><TD style='width:35px;text-align:center;padding-right:10px;'>3<TD style='width:100px;'><A class='noul_link part_link' href='http://parts.igem.org/wiki/index.php?title=Part:BBa_K1342003'>BBa_K1342003</A><TD style='width:80px;'><A class='noul_link' href='http://parts.igem.org/wiki/index.php?title=Part:pSB1C3'>pSB1C3</A><TD style='width:60px;'><TD style='width:90px;'>Accepted<TD style='width:auto;'>Sending Plasmid Backbone<BR><TD style='width:140px;'>&nbsp;</TABLE><DIV style='height: 3px'></DIV>
+<TABLE id='table_contents' width='915' border='0' cellspacing='0' cellpadding='0' style='position:relative;margin-left: 10px;border: 1px solid gray;background-color: #fda; padding-left: 0px;padding-right:2px;padding-top:3px;line-height: 150%; ' >
+<TABLE_BREAK>
+<!-- Subtable for 80803 -->
+<TR><TD style='width:35px;text-align:center;padding-right:10px;'>4<TD style='width:100px;'><A class='noul_link part_link' href='http://parts.igem.org/wiki/index.php?title=Part:BBa_K1342004'>BBa_K1342004</A><TD style='width:80px;'><A class='noul_link' href='http://parts.igem.org/wiki/index.php?title=Part:pSB1C3'>pSB1C3</A><TD style='width:60px;'><TD style='width:90px;'>Accepted<TD style='width:auto;'>Sending Plasmid Backbone<BR><TD style='width:140px;'>&nbsp;</TABLE><DIV style='height: 3px'></DIV>
+<TABLE id='table_contents' width='915' border='0' cellspacing='0' cellpadding='0' style='position:relative;margin-left: 10px;border: 1px solid gray;background-color: #fda; padding-left: 0px;padding-right:2px;padding-top:3px;line-height: 150%; ' >
+<TABLE_BREAK>
+<!-- Subtable for 80804 -->
+<TR><TD style='width:35px;text-align:center;padding-right:10px;'>5<TD style='width:100px;'><A class='noul_link part_link' href='http://parts.igem.org/wiki/index.php?title=Part:BBa_K1342005'>BBa_K1342005</A><TD style='width:80px;'><A class='noul_link' href='http://parts.igem.org/wiki/index.php?title=Part:pSB1C3'>pSB1C3</A><TD style='width:60px;'><TD style='width:90px;'>Accepted<TD style='width:auto;'>Sending Plasmid Backbone<BR><TD style='width:140px;'>&nbsp;
+</TABLE><!-- end content -->