Team:UC-Santa Cruz/Project/Overview

From 2013.igem.org

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1. colony picks.
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<head>
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1.label two microcentrifuge tubes one with “E.Coli+PVCFPC-4+LB+GENT” and the other with   “E.Coli +PXYFPC-2 +LB+KAN ” and make sure to put the date on each tube.
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<meta name=Title content="">
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2. pour 30ml LB into tube labeled “E.Coli+PVCFPC-4+LB+GENT” add Gentamicin to final concentration of 15ug/ml.
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<meta name=Keywords content="">
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3. pour 30ml LB into  tube labeled “E.Coli+PXYFPC-2+LB+KAN” add Kanamycin  to final concentration of 30ug/ml.
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<meta http-equiv=Content-Type content="text/html; charset=macintosh">
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4.take p10 tip , scrape off ONE COLONY off of the plate that reads “LS4260  PXYFPC-2”(in fridge box that says iGEM on it), eject tip into into its respective microcentrifuge tube tube.
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5. repeat with plate that reads “ LS4342 PVCFPC-4”
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6. grow overnight at 37C.
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</head>
-
+
<body bgcolor=white lang=EN-US style='tab-interval:.5in'>
-
2. make electrocompetent CB15N?
+
-
0. grow colony pick overnight CB15N in PYE @ 37C
+
-
1.7? check OD600 of culture
+
-
2?balence culture tubes < 0.01 g difference
+
-
3? spin at 3000 rpm 4C for 15 minutes
+
-
4? discard supernatant
+
-
5?add 10 ml Milli Q water
+
-
6? spin at 3000 rpm 4C for 15 minutes
+
-
7?discard supernatant
+
-
8?repeat step 6 and 7 3 times
+
-
9.? make aliquots and put in deep freezer.
+
-
3.miniprep on ecoli cultures
+
-
1.spin ecoli culture at 3000rpm for 5 minutes
+
-
2.discard supernatant.
+
-
3. resuspend in 6ml of Mili Q water.
+
-
4.add 1ml 6x Lysis buffer, invert 4-6 times, let sit for 5 minutes
+
-
5.add entire mix to blue zymo-midi-filter and  column, place  in 50ml conical tube. spin 5000 rcf for 6 minutes
+
-
6.remove and discard filter and place column on collection tube. Place in benchtop centrifuge at max speed for 30 seconds.
+
-
7.add 400ul endo-wash and spin at max speed on benchtop centrifuge
+
-
8.add 400ul zippy wash buffer , centifuge at max speed for 1 minute, repeat once.
+
-
9. transfer to 1.5 ml epedorf tube add 150ul water, let sit for 1 minute before spinning at max speed for 1 minute
+
-
10. check concentration with nanodrop
+
 +
<div class=Section1>
-
+
<p class=MsoNormal style='margin-bottom:6.0pt;mso-pagination:none;mso-layout-grid-align:
-
3.transfect CB15N with plasmids to make two strains of transformed CB15N.
+
none;text-autospace:none'><span style='font-size:19.0pt;font-family:Helvetica;
-
4.grow strains on plates.
+
mso-bidi-font-family:Helvetica'>ABSTRACT<o:p></o:p></span></p>
-
5.pick colonies for overnights.
+
-
6.induce fluorescence.
+
-
7.check for fluorescence.
+
 +
<p class=MsoNormal style='margin-bottom:6.0pt;mso-pagination:none;mso-layout-grid-align:
 +
none;text-autospace:none'><span style='font-size:13.0pt;font-family:Helvetica;
 +
mso-bidi-font-family:Helvetica'>&#8232;Fresh water shortages affect half of the
 +
world’s population (currently 7 billion persons). In the next 25 years the
 +
numbers of people impacted by <u>severe</u> water shortages is expected to
 +
increase four fold. Shortage of potable water has been estimated to account for
 +
80-90% of disease and 30% of mortality for humanity. <span class=GramE>Roughly
 +
half of all accessible fresh waters (rivers, lakes and underground aquifers)
 +
are estimated to be in use by the current world population</span>. Given only 2.5
 +
percent of the total volume of water is fresh water and this percentage is
 +
shrinking with global warming, fresh water is becoming a critical limiting
 +
resource for human populations. The goal of this project is to develop an
 +
efficient, green, scalable, low cost solution for desalinization of <span
 +
class=GramE>sea water</span> and brackish water. Our solution is creation of a <span
 +
class=SpellE>biofilm</span> (<span class=SpellE>biomachine</span><span
 +
class=GramE>) which</span> uses sunlight to pump sodium chloride across the <span
 +
class=SpellE>biofilm</span> for the desalinization of salt and brackish waters, essentially replacing the pump, membrane, and energy source of a reverse osmosis machine.
 +
The project also provides a scaffold for possible <span class=SpellE>biofilm</span>
 +
cleanup of heavy metals and other toxic ionic pollutants.<o:p></o:p></span></p>
-
Gentamycin Dilution
+
<p class=MsoNormal style='margin-bottom:11.0pt;mso-pagination:none;mso-layout-grid-align:
 +
none;text-autospace:none'><span style='font-size:19.0pt;font-family:Helvetica;
 +
mso-bidi-font-family:Helvetica'>Project Goals<o:p></o:p></span></p>
-
1.weigh out .1g Genamycin powder
+
<p class=MsoListParagraphCxSpFirst style='margin-bottom:11.0pt;mso-add-space:
-
2.place in 50ml microcentrifuge tube
+
auto;text-indent:-.25in;mso-pagination:none;mso-list:l10 level1 lfo11;
-
3.add 10 ml milli q water
+
mso-layout-grid-align:none;text-autospace:none'><![if !supportLists]><span
-
4.pull plunger out of 60ml BD syringe
+
style='mso-bidi-font-size:19.0pt;font-family:Helvetica;mso-fareast-font-family:
-
5.screw on filter tip.
+
Helvetica;mso-bidi-font-family:Helvetica'><span style='mso-list:Ignore'>1.<span
-
6.fill syringe with solution
+
style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp; </span></span></span><![endif]><span
-
7.empty syringe into new 50ml microcentifuge tube.
+
style='mso-bidi-font-size:19.0pt;font-family:Helvetica;mso-bidi-font-family:
 +
Helvetica'>Identify a prototype <span class=SpellE>polarizable</span>
 +
prokaryotic <span class=SpellE>biofilm</span> species<o:p></o:p></span></p>
-
Sterilized colony pick
+
<p class=MsoListParagraphCxSpMiddle style='margin-bottom:11.0pt;mso-add-space:
-
0. autoclave growth media in container. (allow liberal space in container for aeration of culture), after cooling, add appropriate amount of antibiotic.
+
auto;text-indent:-.25in;mso-pagination:none;mso-list:l10 level1 lfo11;
-
1.heat tungsten loop on bunsen burner until red hot.
+
mso-layout-grid-align:none;text-autospace:none'><![if !supportLists]><span
-
2.let cool beside flame
+
style='mso-bidi-font-size:19.0pt;font-family:Helvetica;mso-fareast-font-family:
-
3.dip loop in agarose to check temperature
+
Helvetica;mso-bidi-font-family:Helvetica'><span style='mso-list:Ignore'>2.<span
-
4. beneath flame, carefully scrape-off one colony from plate making sure not to get any other colonies on loop.
+
style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp; </span></span></span><![endif]><span
-
5.beneath flame, stick loop with colony in growth media making sure not to touch the sides of the container with loop. swirl around until colony washes off of loop.
+
style='mso-bidi-font-size:19.0pt;font-family:Helvetica;mso-bidi-font-family:
-
6.incubate in appropriate conditions.
+
Helvetica'>Identify and Clone <span class=GramE>protein tagging</span> peptides
 +
for intracellular targeting of pumps and channels to specific cellular poles.<o:p></o:p></span></p>
-
making LB aggar plates (one liter)
+
<p class=MsoListParagraphCxSpMiddle style='margin-bottom:11.0pt;mso-add-space:
-
1. weight out 150 grams of aggar and add water for LB of 150 mg/ml
+
auto;text-indent:-.25in;mso-pagination:none;mso-list:l10 level1 lfo11;
-
2.autoclave
+
mso-layout-grid-align:none;text-autospace:none'><![if !supportLists]><span
-
3.cool to ~50C
+
style='mso-bidi-font-size:19.0pt;font-family:Helvetica;mso-fareast-font-family:
-
5.add antibiotic
+
Helvetica;mso-bidi-font-family:Helvetica'><span style='mso-list:Ignore'>3.<span
-
6.pour on plates
+
style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp; </span></span></span><![endif]><span
-
7.leave lid of plates slightly off to reduce condensation and contamination.
+
style='mso-bidi-font-size:19.0pt;font-family:Helvetica;mso-bidi-font-family:
-
8.once plates have solidified, stack them with gel facing up
+
Helvetica'>Identify and Clone a series of sunlight driven ion pumps (<span
-
9. place in plastic bag to reduce drying out.
+
class=SpellE>halorhodopsins</span>)<o:p></o:p></span></p>
 +
<p class=MsoListParagraphCxSpMiddle style='margin-bottom:11.0pt;mso-add-space:
 +
auto;text-indent:-.25in;mso-pagination:none;mso-list:l10 level1 lfo11;
 +
mso-layout-grid-align:none;text-autospace:none'><![if !supportLists]><span
 +
style='mso-bidi-font-size:19.0pt;font-family:Helvetica;mso-fareast-font-family:
 +
Helvetica;mso-bidi-font-family:Helvetica'><span style='mso-list:Ignore'>4.<span
 +
style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp; </span></span></span><![endif]><span
 +
style='mso-bidi-font-size:19.0pt;font-family:Helvetica;mso-bidi-font-family:
 +
Helvetica'>Identify and Clone a series of ion channels<o:p></o:p></span></p>
 +
<p class=MsoListParagraphCxSpLast style='margin-bottom:11.0pt;mso-add-space:
 +
auto;text-indent:-.25in;mso-pagination:none;mso-list:l10 level1 lfo11;
 +
mso-layout-grid-align:none;text-autospace:none'><![if !supportLists]><span
 +
style='mso-bidi-font-size:19.0pt;font-family:Helvetica;mso-fareast-font-family:
 +
Helvetica;mso-bidi-font-family:Helvetica'><span style='mso-list:Ignore'>5.<span
 +
style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp; </span></span></span><![endif]><span
 +
style='mso-bidi-font-size:19.0pt;font-family:Helvetica;mso-bidi-font-family:
 +
Helvetica'>Assemble appropriate tag-pump/channel constructs for assessing <span
 +
class=SpellE>NaCl</span> movement across the <span class=SpellE>biofilm</span>.<o:p></o:p></span></p>
-
plasmid miniprep
+
<p class=MsoNormal style='margin-bottom:11.0pt;mso-pagination:none;mso-layout-grid-align:
-
materials
+
none;text-autospace:none'><span style='font-size:19.0pt;font-family:Helvetica;
-
-zippy plasmid miniprep kit
+
mso-bidi-font-family:Helvetica'>Experimental Approach<o:p></o:p></span></p>
-
-50 ml conical tubes
+
-
-1.5ml eppendorf tubes
+
-
-mili Q water
+
-
1. spin down cultures from 9-5-13
+
-
2. discard supernatant
+
-
3.add 6ml of mili q water
+
-
4. resuspend pellets by vortexing
+
-
5.add 1.5 ml 7x lysis buffer, invert tube 4-6 tubes, sit for 5 minutes.
+
-
6.add 3.5 ml of cold Neutralization Buffer (yellow), invert 4-6 times after yellow precipitate starts to form, invert 2-3 times.
+
-
7. place zymo-midi filter/zymo-spin-V-E column assembly into a clean 50 ml conical tube.
+
-
8.add 400ul of endo-wash buffer and centrifuge at 11,000 for 1 minute . Repeat for 1 minute to eliminate residue ( just column without filter)
+
-
9.transfer column into 1.5 ml microcentifuge tube and then add 150ul zippy elution buffer centrifuge for 1 minute after incubation for 1 minute.
+
-
Week 1
+
<p class=MsoListParagraphCxSpFirst style='margin-bottom:6.0pt;mso-add-space:
-
8-1-13
+
auto;text-indent:-.25in;mso-pagination:none;mso-list:l11 level1 lfo12;
-
Made and autoclaved PYE
+
mso-layout-grid-align:none;text-autospace:none'><![if !supportLists]><b
-
prepared another batch of  electrocompetent caulobacter (culture OD600 = 0.615A)
+
style='mso-bidi-font-weight:normal'><span style='font-size:13.0pt;font-family:
-
induced KAN resistant with xylose transformed caulobacter @ OD600 = 0.664A
+
Helvetica;mso-fareast-font-family:Helvetica;mso-bidi-font-family:Helvetica'><span
-
non induced : 3.25ml PXYFPC-2 + CB15N, 20.5ul antibiotic, 200ml PYE
+
style='mso-list:Ignore'>1.<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;
-
induced: 3.25ml PXYFPC-2+ CB15N, 20.5 ul antibiotic, 200ml PYE, .4g xylose .
+
</span></span></span></b><![endif]><b style='mso-bidi-font-weight:normal'><span
-
electroporation of CB15N with PVCFPC-4 .
+
style='mso-bidi-font-size:19.0pt;font-family:Helvetica;mso-bidi-font-family:
-
plated on PYE+agarose+gent plates 5 plates (10ul drop, 50ul drop, 100ul drop, two negative contols (see 8-5-13))
+
Helvetica'>Identify a prototype <span class=SpellE>polarizable</span>
-
plated CB15N on KAN plate for negative control. (note : this was not prepared following the electroporation protocol with water instead of plasmid. This was just a drop from a colony pick shaking incubator pick of CB15N )
+
prokaryotic <span class=SpellE>biofilm</span> species. </span></b><span
-
8-2-13
+
style='font-size:13.0pt;font-family:Helvetica;mso-bidi-font-family:Helvetica'>Our
-
checked fluorescence of PCXFPC-2 : UI (OD600= .76 flouereced, I OD600 = .72 no flourecece.)
+
project aims to create a microbial desalination system using the bacteria <span
 +
class=SpellE><i style='mso-bidi-font-style:normal'>Caulobacter</i></span><i
 +
style='mso-bidi-font-style:normal'> <span class=SpellE>crescentus</span></i>. <span
 +
class=SpellE><i style='mso-bidi-font-style:normal'>Caulobacter</i></span><i
 +
style='mso-bidi-font-style:normal'> <span class=SpellE>crescentus</span></i>
 +
was chosen in our initial experiments for five reasons- 1) it is non-pathogenic
 +
and widely distributed in the fresh water aquatic environment; 2) it is
 +
polarized, making it possible to orient ion pumps and channels on specific sides
 +
(poles) of the bacteria; 3) the stalk allows <span class=SpellE><i
 +
style='mso-bidi-font-style:normal'>Caulobacter</i></span> to attach itself to
 +
solid surfaces where it forms a monolayer of cells (<span class=SpellE>biofilm</span>);
 +
4) a set of plasmids is available for transformation of <span class=SpellE><i
 +
style='mso-bidi-font-style:normal'>Caulobacter</i></span>; and 5) experimental fluorescent
 +
tagged protein models are available for investigation of protein tagging
 +
mechanisms of pole formation (stalk and flagellum) in <span class=SpellE><i
 +
style='mso-bidi-font-style:normal'>Caulobacter</i></span>. Please see the
 +
following key references-<b style='mso-bidi-font-weight:normal'><o:p></o:p></b></span></p>
 +
<p class=MsoListParagraphCxSpMiddle style='margin-bottom:6.0pt;mso-add-space:
 +
auto;mso-pagination:none;mso-layout-grid-align:none;text-autospace:none'><span
 +
class=GramE><span style='mso-bidi-font-size:19.0pt;font-family:Helvetica;
 +
mso-bidi-font-family:Helvetica'>Transcriptional Profiling of <span
 +
class=SpellE>Caulobacter</span> <span class=SpellE>crescentus</span> during
 +
Growth on Complex and Minimal Media.</span></span><span style='mso-bidi-font-size:
 +
19.0pt;font-family:Helvetica;mso-bidi-font-family:Helvetica'> <span
 +
class=SpellE>Hottes</span> et al. Journal of Bacteriology 186(5)<span
 +
class=GramE>:1448</span>-1461(2004).</span><span style='font-size:13.0pt;
 +
font-family:Helvetica;mso-bidi-font-family:Helvetica'><o:p></o:p></span></p>
 +
<p class=MsoListParagraphCxSpMiddle style='margin-bottom:11.0pt;mso-add-space:
 +
auto;text-indent:-.25in;mso-pagination:none;mso-list:l11 level1 lfo12;
 +
mso-layout-grid-align:none;text-autospace:none'><![if !supportLists]><b
 +
style='mso-bidi-font-weight:normal'><span style='mso-bidi-font-size:19.0pt;
 +
font-family:Helvetica;mso-fareast-font-family:Helvetica;mso-bidi-font-family:
 +
Helvetica'><span style='mso-list:Ignore'>2.<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;
 +
</span></span></span></b><![endif]><b style='mso-bidi-font-weight:normal'><span
 +
style='mso-bidi-font-size:19.0pt;font-family:Helvetica;mso-bidi-font-family:
 +
Helvetica'>Identify and Clone <span class=GramE>protein tagging</span> peptides
 +
for intracellular targeting of pumps and channels to specific cellular poles. </span></b><span
 +
style='font-size:13.0pt;font-family:Helvetica;mso-bidi-font-family:Helvetica'>A
 +
series of protein tags have been identified in pole formation in <span
 +
class=SpellE><i style='mso-bidi-font-style:normal'>Caulobacter</i></span>.
 +
These include <span class=SpellE>DivJ</span>, <span class=SpellE>TipF</span>, <span
 +
class=SpellE>StpX</span>, and <span class=SpellE>PflI</span>. Cloning of these
 +
tags are currently underway. Please see the following key references-</span><b
 +
style='mso-bidi-font-weight:normal'><span style='mso-bidi-font-size:19.0pt;
 +
font-family:Helvetica;mso-bidi-font-family:Helvetica'><o:p></o:p></span></b></p>
 +
<p class=MsoListParagraphCxSpMiddle style='margin-bottom:11.0pt;mso-add-space:
 +
auto;mso-pagination:none;mso-layout-grid-align:none;text-autospace:none'><span
 +
class=SpellE><span class=GramE><span style='mso-bidi-font-size:19.0pt;
 +
font-family:Helvetica;mso-bidi-font-family:Helvetica'>PflI</span></span></span><span
 +
class=GramE><span style='mso-bidi-font-size:19.0pt;font-family:Helvetica;
 +
mso-bidi-font-family:Helvetica'>, a Protein Involved in <span class=SpellE>Flagellar</span>
 +
Positioning in <span class=SpellE>Caulobacter</span>.</span></span><span
 +
style='mso-bidi-font-size:19.0pt;font-family:Helvetica;mso-bidi-font-family:
 +
Helvetica'> <span class=SpellE>Obuchowski</span> and Jacobs-Wagner. <i
 +
style='mso-bidi-font-style:normal'>Journal of Bacteriology</i> 190(5)<span
 +
class=GramE>:1718</span>-1729 (2008).<o:p></o:p></span></p>
 +
<p class=MsoListParagraphCxSpMiddle style='margin-bottom:11.0pt;mso-add-space:
 +
auto;mso-pagination:none;mso-layout-grid-align:none;text-autospace:none'><span
 +
class=GramE><span style='mso-bidi-font-size:19.0pt;font-family:Helvetica;
 +
mso-bidi-font-family:Helvetica'>Protein Sequences and Cellular Factors Required
 +
for Polar Localization of <span class=SpellE>Histidine</span> <span
 +
class=SpellE>Kinase</span> in <span class=SpellE><i style='mso-bidi-font-style:
 +
normal'>Caulobacter</i></span><i style='mso-bidi-font-style:normal'> <span
 +
class=SpellE>crescentus</span></i>.</span></span><span style='mso-bidi-font-size:
 +
19.0pt;font-family:Helvetica;mso-bidi-font-family:Helvetica'><span
 +
style="mso-spacerun: yes">&nbsp; </span><span class=SpellE>Sciochetti</span> et
 +
al., Journal of Bacteriology 184(21)<span class=GramE>:6037</span>-6049 (2002).<o:p></o:p></span></p>
-
Week 2
+
<p class=MsoListParagraphCxSpMiddle style='margin-bottom:11.0pt;mso-add-space:
-
8-5-13
+
auto;mso-pagination:none;mso-layout-grid-align:none;text-autospace:none'><span
-
checked plates
+
class=GramE><span style='mso-bidi-font-size:19.0pt;font-family:Helvetica;
-
neg control (CB15N+KAN electroporated with water): one colony
+
mso-bidi-font-family:Helvetica'>Protein localization and <span class=SpellE>dymanics</span>
-
neg control (CB15N+KAN straight from culture): many colonies
+
within a bacterial organelle.</span></span><span style='mso-bidi-font-size:
-
10ul, 50ul, and 100ul many colonies
+
19.0pt;font-family:Helvetica;mso-bidi-font-family:Helvetica'> Hughes et al.
-
plates deemed unusable
+
PNAS<span class=GramE>:107</span>(12):559-5604(2010).<o:p></o:p></span></p>
 +
<p class=MsoListParagraphCxSpMiddle style='margin-bottom:11.0pt;mso-add-space:
 +
auto;text-indent:-.25in;mso-pagination:none;mso-list:l11 level1 lfo12;
 +
mso-layout-grid-align:none;text-autospace:none'><![if !supportLists]><b
 +
style='mso-bidi-font-weight:normal'><span style='mso-bidi-font-size:19.0pt;
 +
font-family:Helvetica;mso-fareast-font-family:Helvetica;mso-bidi-font-family:
 +
Helvetica'><span style='mso-list:Ignore'>3.<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;
 +
</span></span></span></b><![endif]><b style='mso-bidi-font-weight:normal'><span
 +
style='mso-bidi-font-size:19.0pt;font-family:Helvetica;mso-bidi-font-family:
 +
Helvetica'>Identify and Clone a series of sunlight driven ion pumps (<span
 +
class=SpellE>halorhodopsins</span>). </span></b><span style='mso-bidi-font-size:
 +
19.0pt;font-family:Helvetica;mso-bidi-font-family:Helvetica'>Candidate channels
 +
include the <span class=SpellE>halorhodopsins</span> and <span class=SpellE>bacterorhodopsins</span>.
 +
We have cloned one new Na pumping <span class=SpellE>halorhodopsin</span>, KR2,
 +
a protein from a marine <span class=SpellE>flavobacterium</span>, <span
 +
class=SpellE>Krokinobacter</span> <span class=SpellE>eikastus</span>. </span><span
 +
style='font-size:13.0pt;font-family:Helvetica;mso-bidi-font-family:Helvetica'>Please
 +
see the following key reference- <span class=GramE>A</span> light-driven sodium
 +
ion pump in marine bacteria. Inoue et al. Nature Communications DOI: 10.1038/ncomms2689
 +
April 9, 2013. </span><span style='mso-bidi-font-size:19.0pt;font-family:Helvetica;
 +
mso-bidi-font-family:Helvetica'>We are investigating function of BBaK559000 a <span
 +
class=SpellE>BioBrick</span> containing a <span class=SpellE>halorhodopsin</span>.
 +
We are also investigating other sources of <span class=SpellE>halorhodopsins</span>.<b
 +
style='mso-bidi-font-weight:normal'><o:p></o:p></b></span></p>
-
Marshall Porter 8-5-13
+
<p class=MsoListParagraphCxSpMiddle style='margin-bottom:11.0pt;mso-add-space:
-
Made new PYE + Gentamycin plates
+
auto;text-indent:-.25in;mso-pagination:none;mso-list:l11 level1 lfo12;
 +
mso-layout-grid-align:none;text-autospace:none'><![if !supportLists]><b
 +
style='mso-bidi-font-weight:normal'><span style='mso-bidi-font-size:19.0pt;
 +
font-family:Helvetica;mso-fareast-font-family:Helvetica;mso-bidi-font-family:
 +
Helvetica'><span style='mso-list:Ignore'>4.<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;
 +
</span></span></span></b><![endif]><b style='mso-bidi-font-weight:normal'><span
 +
style='mso-bidi-font-size:19.0pt;font-family:Helvetica;mso-bidi-font-family:
 +
Helvetica'>Identify and Clone a series of ion channels. </span></b><span
 +
style='mso-bidi-font-size:19.0pt;font-family:Helvetica;mso-bidi-font-family:
 +
Helvetica'>We have not yet cloned any ion channels. These proteins are our next
 +
goal for gene cloning.<b style='mso-bidi-font-weight:normal'><o:p></o:p></b></span></p>
-
8-6-13
+
<p class=MsoListParagraphCxSpLast style='margin-bottom:11.0pt;mso-add-space:
-
PXYFPC-2 +CB15N culture to be induced made
+
auto;text-indent:-.25in;mso-pagination:none;mso-list:l11 level1 lfo12;
-
-5ml PXYFPC-2 + CB15N (OD600 = 0.72)
+
mso-layout-grid-align:none;text-autospace:none'><![if !supportLists]><b
-
-20ul KAN
+
style='mso-bidi-font-weight:normal'><span style='mso-bidi-font-size:19.0pt;
-
50 ul PCR of stpx and pflI on CB15N
+
font-family:Helvetica;mso-fareast-font-family:Helvetica;mso-bidi-font-family:
-
gel electropheresis on PCR reactions
+
Helvetica'><span style='mso-list:Ignore'>5.<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;
 +
</span></span></span></b><![endif]><b style='mso-bidi-font-weight:normal'><span
 +
style='mso-bidi-font-size:19.0pt;font-family:Helvetica;mso-bidi-font-family:
 +
Helvetica'>Assemble appropriate tag-pump/channel constructs for assessing <span
 +
class=SpellE>NaCl</span> movement across the <span class=SpellE>biofilm</span>.
 +
</span></b><span style='mso-bidi-font-size:19.0pt;font-family:Helvetica;
 +
mso-bidi-font-family:Helvetica'>Work is in progress for creation of
 +
transforming plasmids coding for fusion <span class=GramE>proteins which</span>
 +
include the targeting peptide, the pump or channel and a fluorescent tag. These
 +
plasmids will be used to transform <span class=SpellE><i style='mso-bidi-font-style:
 +
normal'>Caulobacter</i></span><i style='mso-bidi-font-style:normal'>. </i><span
 +
class=GramE>Function will be assessed by determining sub-cellular localization
 +
of the fluorescent tag and assessment for sodium and chloride flux across the <span
 +
class=SpellE>biofilm</span></span>.<b style='mso-bidi-font-weight:normal'><o:p></o:p></b></span></p>
-
8-7-13
+
<p class=MsoNormal style='mso-pagination:none;mso-layout-grid-align:none;
-
transformation of CB15N with PCXFPC-2 (electroporation) time constant = 5.6
+
text-autospace:none'><b><span style='font-size:17.0pt;font-family:Helvetica;
-
transformation of CB15N with PVCFPC-4 (electroporation) time constant = 5.4
+
mso-bidi-font-family:Helvetica'><o:p>&nbsp;</o:p></span></b></p>
-
PXYFPC-2 +CB15N culture to be induced made
+
-
-5ml PXYFPC-2 + CB15N (OD600 = 0.72)
+
-
-20ul KAN
+
-
Marshall Porter 8-7-13
+
<p class=MsoNormal style='margin-bottom:5.0pt;mso-pagination:none;mso-layout-grid-align:
-
● TD PCR for Stpx and pflI tags from caulobacter, two 15 cycle sets
+
none;text-autospace:none'><span style='font-size:17.0pt;font-family:Helvetica;
-
● initial denature of 95 C
+
mso-bidi-font-family:Helvetica;mso-bidi-font-weight:bold'>For Experimental Results,
 +
see: <span style="mso-spacerun: yes">&nbsp; </span><o:p></o:p></span></p>
-
denature anneal elongation denature anneal elongation
+
<p class=MsoNormal style='margin-bottom:5.0pt;mso-pagination:none;mso-layout-grid-align:
-
95 C 56 C 68 C 95 C 49 C 68 C
+
none;text-autospace:none'><span style='font-size:17.0pt;font-family:Helvetica;
-
:10 :15 2:20 :10 :15 2:20
+
mso-bidi-font-family:Helvetica;mso-bidi-font-weight:bold'>Project <o:p></o:p></span></p>
 +
<p class=MsoNormal style='margin-bottom:5.0pt;mso-pagination:none;mso-layout-grid-align:
 +
none;text-autospace:none'><span style='font-size:17.0pt;font-family:Helvetica;
 +
mso-bidi-font-family:Helvetica;mso-bidi-font-weight:bold'>Cell polarization<o:p></o:p></span></p>
-
Gel?...unsucessful
+
<p class=MsoNormal style='margin-bottom:5.0pt;mso-pagination:none;mso-layout-grid-align:
-
Week 3
+
none;text-autospace:none'><span style='font-size:17.0pt;font-family:Helvetica;
-
8-13-13
+
mso-bidi-font-family:Helvetica;mso-bidi-font-weight:bold'>Ion Pumps<o:p></o:p></span></p>
-
TD PCR for Stpx and pflI #3, 4, and 5. Two sets of 15 cycles with initial denature at 95 C for 1:00 for #3 and 94 C for 3:00 for #4 and #5
+
-
#3
+
<p class=MsoNormal style='margin-bottom:5.0pt;mso-pagination:none;mso-layout-grid-align:
-
denature anneal elongation denature anneal elongation
+
none;text-autospace:none'><span class=SpellE><span style='font-size:17.0pt;
-
95 C 56 C 68 C 95 C 49 C 68 C
+
font-family:Helvetica;mso-bidi-font-family:Helvetica;mso-bidi-font-weight:bold'>BioFilm</span></span><span
-
:10 :15 2:20 :15 :15 2:20
+
style='font-size:17.0pt;font-family:Helvetica;mso-bidi-font-family:Helvetica;
 +
mso-bidi-font-weight:bold'><o:p></o:p></span></p>
-
#4
+
<p class=MsoNormal style='margin-bottom:5.0pt;mso-pagination:none;mso-layout-grid-align:
-
denature anneal elongation denature anneal elongation
+
none;text-autospace:none'><span style='font-size:13.0pt;font-family:Helvetica;
-
94 C 56 C 68 C 94 C 49 C 68 C
+
mso-bidi-font-family:Helvetica'><o:p>&nbsp;</o:p></span></p>
-
:15 :30 2:20 :15 :30 2:20
+
-
#5
+
<p class=MsoNormal style='margin-bottom:5.0pt;mso-pagination:none;mso-layout-grid-align:
-
denature anneal elongation denature anneal elongation
+
none;text-autospace:none'><span style='font-size:17.0pt;font-family:Helvetica;
-
94 C 59 C 68 C 94 C 52 C 68 C
+
mso-bidi-font-family:Helvetica;mso-bidi-font-weight:bold'><o:p>&nbsp;</o:p></span></p>
-
:15 :15 2:30 :15 :15 2:30
+
 +
<p class=MsoNormal><o:p>&nbsp;</o:p></p>
-
8-15-13
+
</div>
-
made LB
+
-
checked plates?
+
-
8-19-13
+
-
Gentamicin dilution (10mg/ml)
+
-
1.weigh out .1g genamycin powder
+
-
2.place in 50ml microcentrifuge tube
+
-
3.add 10 ml mili q water
+
-
4.pull plunger out of 60ml BD syringe
+
-
5.screw on filter tip
+
-
6.fill syringe with solution
+
-
7.empty syringe into new 50ml microcentrifuge tube.
+
-
8.aliquot 100ul, 10ul, 500ul, and 10ul into 1.5ml ependorf tubes
+
-
sterilized enoculation of LB with ecoli + PXYFPC-2 +KAN (30ug/ml final concentration)
+
</body>
-
sterilized enocultion of LB with ecoli + PVCFPC-4 +GENT (15ug/ml final concentration)
+
-
8-20-13
+
</html>
-
Glycerol stock of E. coli cultures containing PVCFPC-4
+
Halorhodopsin chloride pump, viewed side-on in the cellular membrane (dotted lines)
-
Plasmid Midi-prep of E. coli cultures
+
[[File:Halorhodopsin.png|400px|center]]
-
● Culture #1 OD550 .916
+
Credit: Michael Kolbe et. al (Science, 2000)  http://opm.phar.umich.edu/
-
● Culture #2 OD5050 2.149
+
-
Nucleic Acid Concentration A260 A280 260/280 260/230
 
-
Culture #1 21.0 ng/ul .419 .221 1.89 1.68
 
-
Culture #2 299.2 ng/ul 5.983 3.184 1.88 2.13
 
-
Week 5
 
-
9-?-13
 
-
use genious to design primers (forward and reverse) to get Halorhodopsin (HsHR) out of H.Salinuarium.
 
-
order primers.
 
-
9-3-13
 
-
take chip of frozen glycerol stock H.Salinarium and thaw in ?
 
-
9-4-13
+
KR2 Rhodopsin sodium pump, viewed side-on, showing ion movement and selected molecular interactions
-
25 ul PCR using HsHR primers ,HS (template), and GC master mix.
+
[[File:RhodopsinKR2.png|400px|center]]
-
negative control without primers labeled (-1)
+
Credit: Keiichi Inoue et. al (Nature 2013)
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negative control without HS (template) labeled (-2)
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98C -2mins/98C 10s 57.2C 72C 2 min/72C 3min hold  4C
+
-
results gel?
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-
 
+
-
 
+
-
 
+
-
9-5-13
+
-
sterilized inoculation of SOC  with iGEM part BBa_K9000 “9000” +ecoli
+
-
sterilized enoculation of SOC with iGEM part BBa_K9001 “9001” +ecoli
+
-
sterilized enoculation of SOC with iGEM part BBa_K9010 “9010” +ecoli
+
-
made chloramphenicol (1ul/1ml) plates
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-
 
+
-
PCR of Halorhodopsin from Halobacterium Salinarum 30 cycles
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25 ul reaction with 2x phusion GC Master Mix
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-
 
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initial denature denature anneal extension final extension
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98 C 98 C 57 C 72 C 72 C
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-
2:00 00:10 00:20 1:00 5:00
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-
 
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-
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9-6-13
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plasmid miniprep on 9000, 9010, and 9010 cultures.
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results
+
-
-9000 = 99.2 ng/ml
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-
-9001 =99.7 ng/ml
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-
-9010 = 38.1 ng/ml
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-
gel of parts
+
-
gell legend : 1Kb ladder , 9010, 9001, 9000
+
-
design and order sequencing primers for iGEM parts. same forward and reverse for all three.
+
-
 
+
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Week 6
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9-9-13
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-
send plasmids with sequencing primers out for sequencing.
+
-
design and order primers for plasmid backbone.
+
-
20ul PCR on HS for HsHR
+
-
run diagnostic gel
+
-
-gel legend : 1Kb ladder , H? , +, -1, -2,
+
-
+
-
 
+
-
 
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-
 
+
-
 
+
-
 
+
-
 
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-
 
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-
 
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-
 
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-
 
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-
 
+
-
 
+
-
 
+
-
 
+
-
 
+
-
 
+
-
 
+
-
9-10-13
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-
50 ul HsHR PCR labeled “50”
+
-
98C 2min/ 98C :10s/57.2C 72 1min/72min/Hold 4C
+
-
 
+
-
 
+
-
+
-
 
+
-
 
+
-
 
+
-
 
+
-
 
+
-
 
+
-
 
+
-
 
+
-
 
+
-
 
+
-
 
+
-
 
+
-
9-11-13
+
-
purification gel
+
-
-gel legend: 1Kb ladder, 50
+
-
+
-
 
+
-
 
+
-
 
+
-
 
+
-
 
+
-
 
+
-
 
+
-
 
+
-
 
+
-
 
+
-
 
+
-
 
+
-
 
+
-
 
+
-
 
+
-
 
+
-
 
+
-
 
+
-
50ul HsHR PCR (two reactions “50(1)” and “50(2)”) one negative control with no template “50 -”
+
-
gel legend : 1Kb ladder , 50(1), 50(2), 50-
+
-
+
-
 
+
-
9-12-13
+
-
50ul PCR to obtain plasmid backbone using 9010 (30.4ng/ul) as template.
+
-
 
+
-
initial denature denature anneal extension final extension
+
-
98 C 98 C 52.2 C 72 C 72 C
+
-
:30 :10 :30 1:00 5:00
+
-
 
+
-
 
+
-
 
+
-
 
+
-
 
+
-
 
+
-
 
+
-
 
+
-
 
+
-
 
+
-
gel legend : 1Kb ladder , 50(1), 50(2), 50-
+
-
+
-
 
+
-
Two 20ul PCR reactions using part 9010 with different annealing temperatures. One at 55 C and one at 57 C
+
-
 
+
-
 
+
-
 
+
-
 
+
-
 
+
-
 
+
-
 
+
-
 
+
-
 
+
-
 
+
-
 
+
-
 
+
-
 
+
-
gel legend : 1Kb ladder , 9010(55 C) , 9010 neg (55 C), 9010 (57 C), 9010 neg (57C)
+
-
 
+
-
+
-
 
+
-
 
+
-
 
+
-
 
+
-
 
+
-
 
+
-
 
+
-
 
+
-
 
+
-
 
+
-
 
+
-
 
+
-
 
+
-
 
+
-
 
+
-
 
+
-
 
+
-
 
+
-
 
+
-
 
+
-
9-14-13
+
-
20ul PCR to obtain plasmid backbone using linearized backbone as template.
+
-
 
+
-
gel legend : 1Kb ladder , 9010 57 C , 9010 57 C(-),  linear 57, linear 57 neg
+
-
+
-
 
+
-
 
+
-
 
+
-
 
+
-
 
+
-
 
+
-
 
+
-
Week 7
+
-
9-15-13
+
-
New PCR of 9010 at 55 C and 57 C with new GC phusion mastermix due to contamination
+
-
and a PCR of Linear PSB1C3 at 57 C
+
-
 
+
-
gel legend : 9010(55+), 9010(55 C-) , 9010(57 C+), 9010(57 C-), Linear (1), Linear (2), Linear(-), DNA ladder
+
-
+
-
 
+
-
9-16-13
+
-
Gel purification on PCR with linearized backbone
+
-
 
+
-
● Run two samples from the PCR reaction, one with 5ul pcr and loading buffer with sybr gold, and the other with the rest of the pcr (45ul) and loading buffer without sybr gold.
+
-
● Load the two samples in side by side lanes
+
-
● Use the sybr gold in uv light to find the band to cut out of the gel in the next lane over.
+
-
● Centrifuge gel cut out with gel filter to separate. Collect filtrate.
+
-
● Use Zymo research DNA Clean and Concentrate to recover DNA from filtrate
+
-
 
+
-
9-17-13
+
-
concentrate DNA
+
-
1.combine all gel purification tubes with purified DNA (add alcohol?)
+
-
2.place sepreate cap with holes in it on top of tube.
+
-
3.place tube in brick under vacuum chamber.
+
-
4.when all liquid is gone, add 5ul of Milli Q water.
+
-
 
+
-
9-18-13
+
-
gibson reaction with KR2 G-blocks
+
-
-5ul concentrated plasmid backbone
+
-
-1ul of each G-block
+
-
-3ul H2O
+
-
-10ul 2x Gibson Master Mix
+
-
-PCR Machine 50C for 1 hr.
+
-
chemicompetent transformation of e.coli with Gibson reaction products.
+
-
plate transformed e.coli on LB+Chloramphenicol plates. (400ul , 200ul, 100ul, and one with untransformed ecoli)
+
-
 
+
-
 
+
-
9-19-13
+
-
suspend colonies in mili q water
+
-
mili q water with colonies colony PCR on colonies from 400ul and 200ul plates
+
-
15 cycles
+
-
94 C initial denature 5 minutes
+
-
94 C denature 15 s
+
-
55 C first anneal 30s
+
-
68 C extension 1:00
+
-
 
+
-
15 cycles anneal drop down 7 C
+
-
94 C denature :15
+
-
48 C second anneal  :30
+
-
68 C extension 1:00
+
-
68 C final extension 5:00
+
-
4 C hold infinite
+
-
 
+
-
inoculation of e. coli colonies in LB + chloramphenicol
+
-
 
+
-
 
+
-
9-20-13
+
-
 
+
-
run gel on PCR products from 9-19-13
+
-
gel legend: 1Kb ladder, “400”, “200”, negative control (no template)
+
-
+
-
 
+
-
Zymo research plasmid midi-prep from e. coli cultures
+
-
9-21-13
+
-
Send psb1c3+KR2 plasmid in for sequencing.
+
-
9-24-13
+
-
receive sequencing results (not sure how to explain the results)
+

Latest revision as of 02:37, 28 September 2013

ABSTRACT


Fresh water shortages affect half of the world’s population (currently 7 billion persons). In the next 25 years the numbers of people impacted by severe water shortages is expected to increase four fold. Shortage of potable water has been estimated to account for 80-90% of disease and 30% of mortality for humanity. Roughly half of all accessible fresh waters (rivers, lakes and underground aquifers) are estimated to be in use by the current world population. Given only 2.5 percent of the total volume of water is fresh water and this percentage is shrinking with global warming, fresh water is becoming a critical limiting resource for human populations. The goal of this project is to develop an efficient, green, scalable, low cost solution for desalinization of sea water and brackish water. Our solution is creation of a biofilm (biomachine) which uses sunlight to pump sodium chloride across the biofilm for the desalinization of salt and brackish waters, essentially replacing the pump, membrane, and energy source of a reverse osmosis machine. The project also provides a scaffold for possible biofilm cleanup of heavy metals and other toxic ionic pollutants.

Project Goals

1.    Identify a prototype polarizable prokaryotic biofilm species

2.    Identify and Clone protein tagging peptides for intracellular targeting of pumps and channels to specific cellular poles.

3.    Identify and Clone a series of sunlight driven ion pumps (halorhodopsins)

4.    Identify and Clone a series of ion channels

5.    Assemble appropriate tag-pump/channel constructs for assessing NaCl movement across the biofilm.

Experimental Approach

1.    Identify a prototype polarizable prokaryotic biofilm species. Our project aims to create a microbial desalination system using the bacteria Caulobacter crescentus. Caulobacter crescentus was chosen in our initial experiments for five reasons- 1) it is non-pathogenic and widely distributed in the fresh water aquatic environment; 2) it is polarized, making it possible to orient ion pumps and channels on specific sides (poles) of the bacteria; 3) the stalk allows Caulobacter to attach itself to solid surfaces where it forms a monolayer of cells (biofilm); 4) a set of plasmids is available for transformation of Caulobacter; and 5) experimental fluorescent tagged protein models are available for investigation of protein tagging mechanisms of pole formation (stalk and flagellum) in Caulobacter. Please see the following key references-

Transcriptional Profiling of Caulobacter crescentus during Growth on Complex and Minimal Media. Hottes et al. Journal of Bacteriology 186(5):1448-1461(2004).

2.    Identify and Clone protein tagging peptides for intracellular targeting of pumps and channels to specific cellular poles. A series of protein tags have been identified in pole formation in Caulobacter. These include DivJ, TipF, StpX, and PflI. Cloning of these tags are currently underway. Please see the following key references-

PflI, a Protein Involved in Flagellar Positioning in Caulobacter. Obuchowski and Jacobs-Wagner. Journal of Bacteriology 190(5):1718-1729 (2008).

Protein Sequences and Cellular Factors Required for Polar Localization of Histidine Kinase in Caulobacter crescentus.  Sciochetti et al., Journal of Bacteriology 184(21):6037-6049 (2002).

Protein localization and dymanics within a bacterial organelle. Hughes et al. PNAS:107(12):559-5604(2010).

3.    Identify and Clone a series of sunlight driven ion pumps (halorhodopsins). Candidate channels include the halorhodopsins and bacterorhodopsins. We have cloned one new Na pumping halorhodopsin, KR2, a protein from a marine flavobacterium, Krokinobacter eikastus. Please see the following key reference- A light-driven sodium ion pump in marine bacteria. Inoue et al. Nature Communications DOI: 10.1038/ncomms2689 April 9, 2013. We are investigating function of BBaK559000 a BioBrick containing a halorhodopsin. We are also investigating other sources of halorhodopsins.

4.    Identify and Clone a series of ion channels. We have not yet cloned any ion channels. These proteins are our next goal for gene cloning.

5.    Assemble appropriate tag-pump/channel constructs for assessing NaCl movement across the biofilm. Work is in progress for creation of transforming plasmids coding for fusion proteins which include the targeting peptide, the pump or channel and a fluorescent tag. These plasmids will be used to transform Caulobacter. Function will be assessed by determining sub-cellular localization of the fluorescent tag and assessment for sodium and chloride flux across the biofilm.

 

For Experimental Results, see:  

Project

Cell polarization

Ion Pumps

BioFilm

 

 

 

Halorhodopsin chloride pump, viewed side-on in the cellular membrane (dotted lines)

Halorhodopsin.png

Credit: Michael Kolbe et. al (Science, 2000) http://opm.phar.umich.edu/


KR2 Rhodopsin sodium pump, viewed side-on, showing ion movement and selected molecular interactions

RhodopsinKR2.png

Credit: Keiichi Inoue et. al (Nature 2013)

Retrieved from "http://2013.igem.org/Team:UC-Santa_Cruz/Project/Overview"