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{{clickpic|iGEM:Cambridge/2008/Notebook/Voltage|igemlogo.gif}}
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'''EmbryoName HK023- chicken embryo- Stage 19.5 - 8.12.14'''




<html>
==Experiment==
<td class="tdboxes"><a href="http://openwetware.org/wiki/iGEM:Cambridge/2008/Notebook/Voltage"><img border="0" src="http://openwetware.org/images/0/00/Vol_logo.JPG" width="256" height="201"></a></td>
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{| {{table}}
'''Objective'''
| align="center" style="background:#f0f0f0;"|''' '''
To transform HB101s with a plasmid encoding GFP (called pGFP) and express this protein by inducing with arabinose.
| align="center" style="background:#f0f0f0;"|''' '''
'''Procedure'''
| align="center" style="background:#f0f0f0;"|'''Personnel'''
| align="center" style="background:#f0f0f0;"|'''Progress'''
|-
| '''Research'''|| ||40 μL||
|-
| ||Potassium intake || ||
|-
| ||Preventing K+ efflux || ||
|-
| ||Bacterial tolerance for high K+ and turgor|| ||
|-
| ||Ligand gated channels || ||
|-
| ||Osmolites (“inert” sugars) || ||
|-
| ||Media || ||
|-
|'''Preliminary wet work ''' || || ||
|-
| ||Extract promoter, RBS and terminator BioBricks from registry
:*Refine protocol for paper-bound DNA extraction
:*Use PCR and transformations to confirm presence of DNA|| ||
|-
| ||Ligand gated channels || ||
|-
|}


 Internal K+ build-up
* Trigeminal nerve bilaterally DiO crystals
o PCR Kdp K+ pump gene from E.coli MG1655
* 7th/8th nerve bilaterally DiI crystals (Big chunk on 7th nerve on L side and big chunk on 8th on R side)
 Design and order primers, including BioBrick prefix and suffix
o Put Kdp gene under control of stationary phase promoter (osmY, used by MIT 2006 team)
 Obtain primer sequences and order, for PCR from BioBricks containing osmY (J45992)
 Ligate to RBS, Kdp gene and terminators in plasmid
o Transform into wildtype and mutant E.coli strains
o Test
 Measure internal K+ concentration using flame photometry


'''Notes'''
No obvious contamination, purposelly put a greater chunk of dye on the 7th nerve on the L side and the 8th nerve on the R side so will see if there is any visible difference between the amount of neurons labelled in different positions within the rhombomere.


 Chassis
==Files==
o Order from Yale
 Kch- mutant, preventing uncontrolled K+ efflux
 Kdp- mutants, preventing regulation of K+ intake
 Kef- mutants, preventing uncontrolled K+ efflux
o Test
 Check competence using YFP BioBrick plasmid
 Measure internal K+ concentration using flame photometry
 Quantify growth relative to wildtype E.coli strain


*
*
*
*


 Controlled K+ efflux
*
o Design sequence based on GluR0 glutamate-gated K+ channel from Synechocystis PCC 6803
**
o Send to DNA 2.0 for synthesis
**
o Backup
 Obtain Synechocystis PCC 6803 strain (from Imperial College London)
 Design and order primers for GluR0, PCR
 Include rare tRNA plasmid in transformation
o Ligate gene into BioBrick plasmid
o Transform into chosen chassis
o Test
 Measure internal K+ concentration with and without presence of glutamate, using flame photometry


==Results==


 Measuring voltage
===Facial===
o Quantify output using oxygen electrode or glass capillary microelectrode
(Green)


* '''''Afferents'''''
 


 Medium optimisation
* '''''Efferents'''''
o Vary K+ concentrations, using KCl
o Vary nutrient levels




 Output optimisation
 
o Vary strength of promoters/RBS
 
 
 
===VIII ===
(green)
 
* '''''Afferents'''''
 
 
 
* '''''Efferents'''''
 
 
* '''''Other'''''
 
 
 
===Basal plate ===
(red)
 
 
==Summary==
 
'''Questions'''
 
'''Future'''
 
 
<META HTTP-EQUIV="refresh" CONTENT="10;URL=http://my.newplace.com/">
 
 
Mary {pad|4em} had a little lamb.
 
'''Badrul Arefin'''
 
 
# Jeong M^, {{spaces|7}} <u>Sun D^</u>, Luo M^, Huang Y, Challen GA, <u>Rodriguez B</u>, Zhang X, Chavez L, Wang H, Hannah R, Kim S, Yang L, Ko M, Chen R, Gottgens B, Lee JS, Gunaratne P, Godley LA, Darlington GJ, Rao A, <u>Li W*</u>, Goodell MA*: Large conserved domains of low DNA methylation maintained by Dnmt3a. <br/> '''''          Nature Genetics''''' . Accepted.
# <u>Akdemir KC</u>, Jain AK, Alton K, Arronow B, J. CA, ..., <u>Li W*</u>, Barton MC*: Genome Wide Profiling Reveals Stimulus-specific Functions of p53 During Differentiation and DNA Damage of Human Embryonic Stem Cells. <br/>'''''Nucleic Acids Res.''''' Accepted
# Klein JB, Piao L, …, <u>Li W</u>, Shi X, Kutateladze TG: KDM5B demethylase binds to its histone target and the product through distinct modules.<br/>''Cell Reports''. Accepted. <br/><br/>'''2013''' <br/><br/>
# <u>Chen K</u>, <u>Xi Y</u>, Pan X, Dent S, He X, <u>Li W*</u>: DANPOS: Dynamic Analysis of Nucleosome Position and Occupancy by Sequencing. <br/>'''''Genome Res.''''' 23 (2013) 341-351. [http://www.bcm.edu/news/item.cfm?newsID=6723 BCM News];  [http://epigenie.com/danpos-reveals-dynamic-nucleosomes Epigenie Headline]  <br/><br/>
# <u>Wang L</u>, <u>Park JH</u>, Wang S, Kocher JP, <u>Li W*</u>: CPAT: Coding-Potential Assessment Tool Using an Alignment-Free Logistic Regression Model. <br/>'''''Nucleic Acids Res.''''' 41 (2013) e74.<br/><br/>
# <u>Lin X</u>, <u>Sun D</u>, <u>Rodriguez B</u>, Zhao Q, Sun H, Zhang Y, <u>Li W*</u>: BseQC: Quality Control of bisulfite sequencing experiments. <br/>'''''Bioinformatics''''' 29 (2013) 3227-3229. PMCID: PMC3842756 .<br/><br/>
# <u>Chen K^</u>, Wilson MA^, Hirsch C, Watson A, Liang S, Lu Y, <u>Li W*</u>, Dent S*: Stabilization of the Promoter Nucleosomes in Nucleosome Free Region by the Yeast Ssn6-Tup1 Corepressor. <br/>'''''Genome Res.''''' 23 (2013) 312-322. <br/><br/>

Revision as of 13:00, 21 December 2014

EmbryoName HK023- chicken embryo- Stage 19.5 - 8.12.14


Experiment

Objective To transform HB101s with a plasmid encoding GFP (called pGFP) and express this protein by inducing with arabinose. Procedure

  • Trigeminal nerve bilaterally DiO crystals
  • 7th/8th nerve bilaterally DiI crystals (Big chunk on 7th nerve on L side and big chunk on 8th on R side)

Notes No obvious contamination, purposelly put a greater chunk of dye on the 7th nerve on the L side and the 8th nerve on the R side so will see if there is any visible difference between the amount of neurons labelled in different positions within the rhombomere.

Files

Results

Facial

(Green)

  • Afferents


  • Efferents




VIII

(green)

  • Afferents


  • Efferents


  • Other


Basal plate

(red)


Summary

Questions

Future


<META HTTP-EQUIV="refresh" CONTENT="10;URL=http://my.newplace.com/">


Mary {pad|4em} had a little lamb.

Badrul Arefin


  1. Jeong M^, Template:Spaces Sun D^, Luo M^, Huang Y, Challen GA, Rodriguez B, Zhang X, Chavez L, Wang H, Hannah R, Kim S, Yang L, Ko M, Chen R, Gottgens B, Lee JS, Gunaratne P, Godley LA, Darlington GJ, Rao A, Li W*, Goodell MA*: Large conserved domains of low DNA methylation maintained by Dnmt3a.
    Nature Genetics . Accepted.
  2. Akdemir KC, Jain AK, Alton K, Arronow B, J. CA, ..., Li W*, Barton MC*: Genome Wide Profiling Reveals Stimulus-specific Functions of p53 During Differentiation and DNA Damage of Human Embryonic Stem Cells.
    Nucleic Acids Res. Accepted
  3. Klein JB, Piao L, …, Li W, Shi X, Kutateladze TG: KDM5B demethylase binds to its histone target and the product through distinct modules.
    Cell Reports. Accepted.

    2013

  4. Chen K, Xi Y, Pan X, Dent S, He X, Li W*: DANPOS: Dynamic Analysis of Nucleosome Position and Occupancy by Sequencing.
    Genome Res. 23 (2013) 341-351. BCM News; Epigenie Headline

  5. Wang L, Park JH, Wang S, Kocher JP, Li W*: CPAT: Coding-Potential Assessment Tool Using an Alignment-Free Logistic Regression Model.
    Nucleic Acids Res. 41 (2013) e74.

  6. Lin X, Sun D, Rodriguez B, Zhao Q, Sun H, Zhang Y, Li W*: BseQC: Quality Control of bisulfite sequencing experiments.
    Bioinformatics 29 (2013) 3227-3229. PMCID: PMC3842756 .

  7. Chen K^, Wilson MA^, Hirsch C, Watson A, Liang S, Lu Y, Li W*, Dent S*: Stabilization of the Promoter Nucleosomes in Nucleosome Free Region by the Yeast Ssn6-Tup1 Corepressor.
    Genome Res. 23 (2013) 312-322.

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