Difference between revisions of "IGEM:Cambridge/2008/Notebook/Voltage"

From OpenWetWare
Jump to: navigation, search
(10 intermediate revisions by 2 users not shown)
Line 33: Line 33:
 
*To simplify the concept, we are only regulating and measuring the flux of potassium (K+) ions, and we are using a directly glutamate-gated K+ ion channel.  
 
*To simplify the concept, we are only regulating and measuring the flux of potassium (K+) ions, and we are using a directly glutamate-gated K+ ion channel.  
 
*This means that on the binding of glutamate, the channels will open, allowing a K+ flux, which will change the voltage of the medium enough to be detected with a very sensitive electrode.
 
*This means that on the binding of glutamate, the channels will open, allowing a K+ flux, which will change the voltage of the medium enough to be detected with a very sensitive electrode.
*In order to set up a large enough K+ concentration gradient across the membrane for ions to flow down when the channels open, an ion pump is necessary. E.coli has a transmembrane P-type ATPase called Kdp, which pumps K+ into the cell. We have isolated this gene to overexpress it, therefore causing the cells to pump in a large number of K+ ions.
+
*In order to set up a large enough K+ concentration gradient across the membrane for ions to flow down when the channels open, cells are grown in high K+ medium (100mM) and resuspended in low K+ medium.
*However, E.coli also has a number of osmoregulatory systems which use relative K+ ion concentrations to control turgor. There are K+ leak channels (Kch and Kef) in the membrane, so we have ordered E.coli strains with mutations in these genes to allow K+ to remain sequestered inside the cells until the glutamate-gated channels open.
+
*However, E.coli also has a number of osmoregulatory systems which use relative K+ ion concentrations to control turgor. There are K+ leak channels (Kch and Kef) in the membrane, so we have chosen E.coli strains with mutations in these genes as our chassis.
  
[[Media:Voltage_project.ppt |Presentation]]
 
  
=Progress=
+
=Experiment Summaries=
  
[[IGEM:Cambridge/2008/Notebook/Voltage/Progress |Progress]]
+
[[IGEM:Cambridge/2008/Notebook/Voltage/Output| Electrical Output]]
 
 
=Experiment Summaries=
 
  
 
[[IGEM:Cambridge/2008/Notebook/Voltage/K+ Growth|Mutant Growth Rates]]
 
[[IGEM:Cambridge/2008/Notebook/Voltage/K+ Growth|Mutant Growth Rates]]
Line 48: Line 45:
 
[[IGEM:Cambridge/2008/Notebook/Voltage/K+ Concentrations|Cytoplasmic K+ Concentrations]]
 
[[IGEM:Cambridge/2008/Notebook/Voltage/K+ Concentrations|Cytoplasmic K+ Concentrations]]
  
[[IGEM:Cambridge/2008/Notebook/Voltage/BioBrick Manipulation|BioBrick Manipulation]]
+
 
 +
Parts Construction:
 +
 
 +
*[[IGEM:Cambridge/2008/Notebook/Voltage/BioBrick Manipulation|KDP]]
 +
 
 +
*[[IGEM:Cambridge/2008/Notebook/Voltage/GluR0 Manipulation|GluR0]]
  
 
*[[IGEM:Cambridge/2008/Extracted_Parts | Extracted Biobrick Parts]]
 
*[[IGEM:Cambridge/2008/Extracted_Parts | Extracted Biobrick Parts]]
 +
 +
 +
 +
=Progress=
 +
 +
[[IGEM:Cambridge/2008/Notebook/Voltage/Progress |Progress]]
  
  
Line 69: Line 77:
  
 
[http://www.uniprot.org/ Uniprot database]
 
[http://www.uniprot.org/ Uniprot database]
 +
 +
[[Media:Voltage_project.ppt |Presentation]]
  
 
=Literature=
 
=Literature=

Revision as of 06:38, 28 October 2008



<!-- sibboleth --><div id="lncal1" style="border:0px;"><div style="display:none;" id="id">lncal1</div><div style="display:none;" id="dtext">07/15/2008,07/16/2008,07/17/2008,07/18/2008,07/21/2008,07/22/2008,07/23/2008,07/24/2008,07/25/2008,07/28/2008,07/29/2008,07/30/2008,07/31/2008,08/01/2008,08/04/2008,08/05/2008,08/06/2008,08/07/2008,08/08/2008,08/11/2008,08/12/2008,08/13/2008,08/14/2008,08/15/2008,08/18/2008,08/19/2008,08/20/2008,08/21/2008,08/22/2008,08/25/2008,08/26/2008,08/27/2008,08/28/2008,08/29/2008,09/02/2008,09/03/2008,09/04/2008,09/05/2008,09/08/2008,09/09/2008,09/10/2008,09/11/2008,09/12/2008,09/15/2008,09/16/2008,09/17/2008,09/18/2008,09/19/2008,09/22/2008,09/23/2008,09/24/2008,09/25/2008,09/26/2008,09/29/2008,09/30/2008</div><div style="display:none;" id="page">IGEM:Cambridge/2008/Notebook/Voltage</div><div style="display:none;" id="fmt">yyyy/MM/dd</div><div style="display:none;" id="css">OWWNB</div><div style="display:none;" id="month"></div><div style="display:none;" id="year"></div><div style="display:none;" id="readonly">Y</div></div>

Voltage button.gif <sitesearch></sitesearch>

Customize your entry pages <html><img src="/images/a/aa/Help.png" border="0" /></html>

Background

  • The voltage output part of our project aims to mimic the signal transduction that occurs at a neural synapse.
  • We are engineering E.coli to create a voltage output on detection of glutamate. This imitates the creation of a postsynaptic potential in a dendrite when a neurotransmitter (such as glutamate) is present at the synapse.
  • The mechanism we have designed is similar to that used in the brain – relying on ion movement across the membrane, and gated ion channels.
  • To simplify the concept, we are only regulating and measuring the flux of potassium (K+) ions, and we are using a directly glutamate-gated K+ ion channel.
  • This means that on the binding of glutamate, the channels will open, allowing a K+ flux, which will change the voltage of the medium enough to be detected with a very sensitive electrode.
  • In order to set up a large enough K+ concentration gradient across the membrane for ions to flow down when the channels open, cells are grown in high K+ medium (100mM) and resuspended in low K+ medium.
  • However, E.coli also has a number of osmoregulatory systems which use relative K+ ion concentrations to control turgor. There are K+ leak channels (Kch and Kef) in the membrane, so we have chosen E.coli strains with mutations in these genes as our chassis.


Experiment Summaries

Electrical Output

Mutant Growth Rates

Cytoplasmic K+ Concentrations


Parts Construction:


Progress

Progress


Technical Information

Gene Design

Flame Photometer Calibration

OD600 (Cell Density) Calibration

Mutant Strains Information


Useful Links

Protein prediction tools

Uniprot database

Presentation

Literature

Kdp operon diagram

plasmid

The Kdp-ATPase system and its regulation

Potential Chassis: |Strain JW1242-1 Strain JW0710-1

Kdp mutant - paper from 1971

Worldwide E.coli Databases

Characterisation of kdpD - 2005

Investigations on Kdp Operon exp. & flux

Very interesting 2001 paper concerning Glutamate Channels

1999 paper on functional characterization of prokaryote Glu Channels

Sequenced Synechocystis PCC 6803 genome

Glutamate-gated K+ channel GluR0

Link to E.coli statistics page (CCDB Database)

Recent changes

26 September 2017

     04:06 

Butlin:Unix for Bioinformatics - advanced tutorial‎‎ (3 changes | history) . . (+334). . [Claudius E Kerth‎ (3×)]

     

04:06

(cur | prev) . . (+148). . Claudius E Kerth (talk | contribs) (Added troublehooting tip: when rename.pl download link does not work, go to my Dropbox)

     

02:44

(cur | prev) . . (+168). . Claudius E Kerth (talk | contribs) (added Troubleshooting tip)

 m   

02:36

(cur | prev) . . (+18). . Claudius E Kerth (talk | contribs) (TASK 5: I have split my reads by barcode and I have quality filtered them. Now I want to know how many reads I have left from each (barcoded) individual. How can I find that out?)

25 September 2017

     21:19 

BME100 f2017:Group12 W0800 L3‎‎ (12 changes | history) . . (+3,361). . [Priscilla Y. Han‎ (12×)]

     

21:19

(cur | prev) . . (+25). . Priscilla Y. Han (talk | contribs) (Descriptive Stats and Graph)

     

21:15

(cur | prev) . . (+70). . Priscilla Y. Han (talk | contribs) (Descriptive Stats and Graph)

     

21:11

(cur | prev) . . (-3). . Priscilla Y. Han (talk | contribs) (Experimental Design of Device)

     

21:10

(cur | prev) . . (-12). . Priscilla Y. Han (talk | contribs) (Experimental Design of Device)

     

21:10

(cur | prev) . . (+15). . Priscilla Y. Han (talk | contribs) (Experimental Design of Device)

     

21:09

(cur | prev) . . (+3,181). . Priscilla Y. Han (talk | contribs) (Experimental Design of Device)

     

21:05

(cur | prev) . . (+5). . Priscilla Y. Han (talk | contribs) (Descriptive Stats and Graph)

     

21:03

(cur | prev) . . (+25). . Priscilla Y. Han (talk | contribs) (Descriptive Stats and Graph)

     

21:01

(cur | prev) . . (+2). . Priscilla Y. Han (talk | contribs) (Descriptive Stats and Graph)

     

20:58

(cur | prev) . . (+29). . Priscilla Y. Han (talk | contribs) (Descriptive Stats and Graph)

     

20:57

(cur | prev) . . (-3). . Priscilla Y. Han (talk | contribs) (Descriptive Stats and Graph)

     

20:55

(cur | prev) . . (+27). . Priscilla Y. Han (talk | contribs) (Descriptive Stats and Graph)

     21:19 

(Upload log). .

[Ana C. Carr‎ (4×); Priscilla Y. Han‎ (7×)]

     

21:19

. . Priscilla Y. Han (talk | contribs) uploaded File:Pulsingbargraph12.png

     

21:18

. . Priscilla Y. Han (talk | contribs) uploaded File:Mypear sonheart12.png

     

21:18

. . Priscilla Y. Han (talk | contribs) uploaded File:Heartratespree12.png

     

21:04

. . Priscilla Y. Han (talk | contribs) uploaded File:Scrnshotspree12.png

     

20:59

. . Priscilla Y. Han (talk | contribs) uploaded File:2columnbarsspree.png

     

20:56

. . Priscilla Y. Han (talk | contribs) uploaded a new version of File:Mypear sonr.png

     

20:56

. . Priscilla Y. Han (talk | contribs) uploaded File:Mypear sonr.png

     

18:36

. . Ana C. Carr (talk | contribs) uploaded a new version of File:IMG 5562.jpg(In action)

     

18:36

. . Ana C. Carr (talk | contribs) uploaded a new version of File:IMG 5562.jpg(In action)

     

18:36

. . Ana C. Carr (talk | contribs) uploaded a new version of File:IMG 5562.jpg(In action)

     

18:36

. . Ana C. Carr (talk | contribs) uploaded a new version of File:IMG 5562.jpg(In action)

     20:33 

BME100 f2017:Group8 W1030 L3‎‎ (9 changes | history) . . (+501). . [Jennifer L. Brodsky‎ (9×)]

     

20:33

(cur | prev) . . (+5). . Jennifer L. Brodsky (talk | contribs) (Experimental Design of Own Device)

     

20:32

(cur | prev) . . (+112). . Jennifer L. Brodsky (talk | contribs) (Experimental Design of Own Device)

     

20:29

(cur | prev) . . (0). . Jennifer L. Brodsky (talk | contribs) (Experimental Design of Own Device)

     

20:28

(cur | prev) . . (+13). . Jennifer L. Brodsky (talk | contribs) (Experimental Design of Own Device)

     

20:28

(cur | prev) . . (-1). . Jennifer L. Brodsky (talk | contribs) (Experimental Design of Own Device)

     

20:26

(cur | prev) . . (+1). . Jennifer L. Brodsky (talk | contribs) (Experimental Design of Own Device)

     

20:26

(cur | prev) . . (+177). . Jennifer L. Brodsky (talk | contribs) (Experimental Design of Own Device)

     

20:25

(cur | prev) . . (+7). . Jennifer L. Brodsky (talk | contribs) (Experimental Design of Own Device)

     

20:24

(cur | prev) . . (+187). . Jennifer L. Brodsky (talk | contribs) (Experimental Design of Own Device)

N    20:17  BME100 f2017:Group10 W0800 L3‎ (diff | hist) . . (+767). . Whitney Hirano (talk | contribs) (Created page with "5. The mean value of heart rate calculated from the pulse ox was 98.3673 beats/min with a standard deviation of 22.2273, while the mean value of heart rate calculated from the...")
     19:56  BME100 f2017:Group9 W0800 L3‎ (diff | hist) . . (+3). . Irene Zhang (talk | contribs) (LAB 3 WRITE-UP)

     19:52 

BME100 f2017:Projects1‎‎ (3 changes | history) . . (+19). . [Yar‎; Karmella Haynes‎ (2×)]

     

19:52

(cur | prev) . . (0). . Yar (talk | contribs) (small change to force cache purge)

     

19:37

(cur | prev) . . (+21). . Karmella Haynes (talk | contribs)

     

19:11

(cur | prev) . . (-2). . Karmella Haynes (talk | contribs) (Lab 1: “Needs Identification”)

     19:19  CONJ606:Schedule‎ (diff | hist) . . (+23). . Maureen E. Hoatlin (talk | contribs) (Week-by-Week Schedule Summary)

     19:06 

BME100 f2017‎‎ (2 changes | history) . . (+1). . [Karmella Haynes‎ (2×)]

     

19:06

(cur | prev) . . (-8). . Karmella Haynes (talk | contribs)

     

19:06

(cur | prev) . . (+9). . Karmella Haynes (talk | contribs)

     17:38  User:Oww Bot‎ (diff | hist) . . (-956). . Yar (talk | contribs) (it me)
     17:36 (User rights log) . . Yar (talk | contribs) changed group membership for Oww Bot from bot and administrator to bot ‎
     17:35  User:Yar‎ (diff | hist) . . (+26). . Yar (talk | contribs) (my name)

     17:34 

(Move log). .

[Yar‎ (3×)]

     

17:34

. . Yar (talk | contribs) moved page User talk:Yardena Cohen to User talk:Yar(Automatically moved page while renaming the user "Yardena Cohen" to "Yar")

     

17:34

. . Yar (talk | contribs) moved page User:Yardena Cohen/Notebook/attempt/Entry Base to User:Yar/Notebook/attempt/Entry Base(Automatically moved page while renaming the user "Yardena Cohen" to "Yar")

     

17:34

. . Yar (talk | contribs) moved page User:Yardena Cohen/Notebook/attempt to User:Yar/Notebook/attempt(Automatically moved page while renaming the user "Yardena Cohen" to "Yar")

     17:34 (User rename log) . . Yar (talk | contribs) renamed user Yardena Cohen (108 edits) to Yar(shorter)