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

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)

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

• The petri dishes we use all the time are made of untreated polystyrene: http://www.bdbiosciences.com/ptProduct.jsp?prodId=363308