IGEM:MIT/2007/Notebook/2007-8-17

Agenda

 * 1) Transform R0051 into BL21
 * 2) Transform last night's ligations (F+B, I.B34.CPX.B14) into BL21
 * 3) Plate transformants
 * 4) Send mer operon PCR products (merR and Pmer) to sequence

Meeting Notes
Polystyrene binding working
 * replacing AHL inducible promorter with constutive promoter
 * ran a western, presented results
 * increasing AHL, more protein (issues loading); thinking put in set amounts of known protein
 * discussion about t7 and its 2 bands, uncertainty why 2 bands
 * could be multimers (perhaps not a worry if it's working) could be modification of the protein
 * '''double check t7 control
 * '''have to put stop codon translational stops (could be read through)
 * '''do finer material in bulk

Mercury Binding received ec20 in mail with lppompa determining hg2+, mass spec won't work use icp-aes instead with half day training session order hg standards samples $30/hr to use seemed fine with 1ppb to 1ppm
 * might run komasi stain if expression of lpp-ompa to find concentration which is iptg inducible
 * '''look up icp-aes works

Mercury Sensor
 * 1) 3A of I13500, mer operon, iat3 - failed
 * 2) mercury binding assay - failed
 * 3) pcr -first pcr worked, second pcr with same primers failed (shouldn't work acc. to tk)
 * 4) gel - failed 600 bp in pcr product and - control, then new one no bands


 * '''annealing temp?
 * '''try again. negative control should be no template

Review of overall project

Things need to be completed -replace iptg operon by mer operon so hg inducible -mer operon mission critical


 * '''many of e coli dna remnants are in lab equipment (like primers, etc) might not even need plasmid
 * '''needs crisp benchwork
 * possible parallel efforts or drag in grads etc