Team:Yale/Notebook/Week10

From 2011.igem.org

Notebook: Week 5

Friday - Planned/mapped out next 4 weeks - Took dialyzed bag with purified eGFP-RiAFP and divided it into 12 eppendorfs, containing 1 mL each. Flash-froze tubes in liquid nitrogen, then quickly used a hot edge to poke holes into top of eppendorfs to allow sublimation. Tubes then placed in lyophilizer for 3 nights.

Saturday - Began cloning all pSB1AK8 constructs into pSB1C3 for parts submission (G3, H1, RiA, RiG, T2, Z2?): - Digested 6 constructs and linearized pSB1C3 with EcoRI, PstI - Also, PCR amplified eGFP segment from original eGFP-RiAFP plasmid to use as a control for survival assays - Digested PCR fragment with eGFP with XbaI, PstI, and digested BBa_I712074 (strong T7 promoter) with SpeI and PstI - Gel extracted all digested pSB1AK8 constructs using kit, gel visualized with SYBRSafe (digestion seemed successful)

Sunday - Ligated gel-extracted pSB1AK8 fragments with pSB1C3; ligated eGFP PCR fragment with BBa_I712074 - Transformed ligations into DH5alpha cells, plated them

Monday - Ran colony PCR on transformed samples; seems to confirm successful ligation (see pics below): - Picked up lyophilized cells; resuspended first in 50 mL of 50 mM Tris-HCl (pH 7.5); A280 ~ .72, A488 ~ .13; however, looked cloudy (possible aggregation?), so centrifuged at 13K rpm for 10 minutes, and UV-vised the supernatant, A280 ~ .23, A488 ~ .04, suggesting around 75% was aggregated. Tried again to resuspend in 100 mL Tris-HCl and adding 50 mM NaCl; still no good; tried adding acid and killed protein activity

Tuesday - Miniprepped successful colony PCRs and submitted samples for sequencing - Prepared more buffers for protein purification - Transformed eGFP and eGFP-RiAFP and RiAFP cells

Wednesday - Protein purification round 2! - Prepared a lysis buffer of 50 mM NaH2PO4, 300 mM NaCl (with 1 mM PMSF added before lysis), and 500 mM imidazole elution buffer (with same salts as lysis buffer) - Innoculated eGFP, eGFP-RiAFP, and RiAFP cells - Resuspended 1 L (500 mL) worth of eGFP-RiAFP (RiAFP) pellets in around 35 mL lysis buffer - Sonicated (using Spiegel lab sonicator) with 1 s on/4 s off for 25 total minutes of sonication for each resuspended pellet (unforunately, RiAFP sample by itself was foaming when we returned...) - Equilibrated 2 1 mL packed Ni-NTA columns (from Modis lab) with lysis buffer using peristaltic pump - Spin-concentrated samples using - Ran w FPLC/Fraction Collector Ni-NTA

Thursday - Ran/checked gels with lots of protein - Grew up more cells - Size exclusion (small load) with eGFP-RiAFP and DTT'ed (after spin concentration) - Incubated with TEV

Friday - Size exclusion of TEV-ed protein -