Team:Bielefeld-CeBiTec/Notebook/CFPS
Cell-free Protein Synthesis
04/22: Transformation of electrocompetent E. coli KRX with BBa_K1365020 (sfGFP (Bs))
04/23: preparing glycerol stock of KRX with BBa_K1365020
04/24-04/25: Mini-prep of BBa_K1365020
04/28: Transformation of electrocompetent E. coli KRX with BBa_I746909 (T7-promoter, RBS, sfGFP, double terminator). Before plating on LB(Cm), 50 µl of 200 g∙L-1 Rhamnose-solution is added to the plate to induce T7-Polymerase. Inoculation of 10 ml preculture of simple KRX from plate. Overnight culturing, shaking vigorously at 220 rpm.
04/29: colonies of KRX with BBa_I746909 look greenish! 2.2 ml Overnight cultures from KRX are used to inoculate 100 ml for start OD600=0.144. Measuring of growth curves for a first estimation of appropriate moment for cell harvest (see image below)
Growth curves (duplicate) from E. coli KRX at 37 °C in 100 ml 2xTY+P-medium. 1 l shaking flasks without baffles were used. Shaking at 200 rpm.
04/30: An aliquot of ER2566 also known as "T7 Express Competent E. coli (High Efficiency)" from NEB was kindly provided by Fabian. Cells were plated on LB.
Plasmid-mini-prep from 3 ml of overnight culture ( E. coli KRX with BBa_I746909), two glycerol stocks prepared.
Preparation of amino acid stock solutions for CFPS acccording to protocol from Caschera and Noireaux 2015. First, all aminoacids were seperatly weighed into microcentrifuge tubes, which took a while. Then, 500 µl of 5 M KOH was added to each amino acid. Solubilization was achieved via multiple inverting and, if necessary, vortexing. Especially tyrosine takes a while, and is a suspension rather than a solution. Stock solutions are afterwards stored at -20 °C. Note: According to Caschera and Noireaux, these stock solutions can only be stored a few weeks.