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 Xis it same scale across all images or does program chooses intensity. eric answered this
 * scale bar for pictures
 * bright+fluorescent
 * 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:<all of energy goes into copying; 10x yield on low copy plasmids. (must check if not chloramphenicol resistant)
 * Possible leads on psb3k3 purifications:
 * let tk know; can watch
 * do chlorampheticol amplification on plasmid
 * 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

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

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