IGEM:MIT/2007/Notebook/2007-8-7

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===For Future (Plate) Polystyrene Assays===
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*The petri dishes we use all the time are made of untreated polystyrene: http://www.bdbiosciences.com/ptProduct.jsp?prodId=363308

Current revision

Contents

Agenda

  1. Transform last night's ligation into DH5a
  2. Plate
  3. Streak cpx-construct cells in preparation for western blot
  4. Group Meeting at 2 in 674

Transformed Ligation product of Standard Assemblies (I14032+B0034, R0051+B0034, CPX+B0014)

Plated 150µl and spun down remainder, centrifuged, resuspended concentrated pellet and plated that as well (pellet was small)

Streaked CPX BL21 cells for Second Western Blot

Meeting Minutes

update
Talked about polystyrene binding-works
Mercury binding-ordered
mercury sensing-not working

compare random sequence to e coli genome
then make part from it

talked about polystyrene assay 
*need to do negative control of (no plasmid)
*check specificity and try polypropylene etc
*in future, arrange results in order of increasing concentrations
*do viable count of bacteria at high concentrations (ask grads about this)

T7 fluorescent antibody tag
*scale bar for pictures
*bright+fluorescent
Xis it same scale across all images or does program chooses intensity. eric answered this
*Future controls: "induce" cells without plasmid. stain with only second antibody.  
*hi res of stained antibody-can we tell localized binding (to each other) or to polystyrene?

what's next:
we've done in pbs, but need to do in water
need to put cpx downstream of promoter

metal binding
using ec20 on lpp-ompa
*find out how well iron binds to ec20
*find out toxic levels for similar metals (iron, zinc).  do background check on this

Mike Bennie:<<ask him
biobricking beta c term domain of n terminus
~1 week away; most pieces are done.

MTA with de Lorenzo.
protein export tag
fos gune??
leucine zipper
beta portion of 84
ege1 has protein autotransporter

*Possible leads on psb3k3 purifications:
*let tk know; can watch
*do chlorampheticol amplification on plasmid
grow overnight, dilute, regrow in chlorampheticol; inhibits protein production but not dna replication->all of energy goes into copying; 10x yield on low copy plasmids. (must check if not chloramphenicol resistant)
*use TE instead of water (trys-EDTA, trys is a buffer, EDTA chelates magnesium, a cofactor of DNase)
*can heat it; if heat elution buffer to 50 or 60 degrees,
*elute by 30 ul twice, then combine

*what percentage of bacteria is binding?
*try fine particulate polystyrene

**engineering fish to prevent mercury acculumation

**nitrocellulose sandwiches
*robbie's a pro at this stuff
robbie or rana for western blot

thurs 10 am 4-261

For Future (Plate) Polystyrene Assays

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