Harvard BioDesign/18 July 2014

From 2014.igem.org

Picked colonies from pHBD45, pHBD1 and pHBD2 transformations from previous day. Mini-prepped pHBD45

Ran PCR fragments for the gibson of pHBD40 from previous day on a gel, gel extracted, gibsoned, and transformed into MACH1 cells. Plated transformation.

Want to grow up cells expressing CsgA to try purification of CsgA and SDS gel runs on Monday. Since we are growing up LSR10 cells with pHBD1 for cryostocks, we will plate some of that culture onto YESCA+Congo Red plates to grow up at 25C over the weekend.

Received ordered oligonucleotides Oligo/primer 1: 30 base pair homology with linker, 39 base pairs of SpyTag, Oligo/primer 2: 30 base pair homology with the pBbE1a backbone, reverse complement of the 39 base pairs of Spy Tag PCR’ed primers/oligos Oligos were primers to each other Thermocycler Repeat 37 times 98 degrees → 15 seconds 72 degrees → 15 seconds Final cool down 72 degrees → 30 seconds Ran a gel with the extended primers/oligos and with the F48 backbone

Cleaned the oligos and F48 backbone Because of leftover salt from the PCR reaction This disrupts the Gibson assembly

Gibson assembly Used Spy Tag as insert and F48 backbone as template

• Read Sofia’s paper: gel purification, testing SpyTag and catcher complex with curli

Transformed CsgA with Spy Tag into Mach I cells Grew up and plates Mach I cells

Super resolution microscopy TEM- Bom SEM- Bom See scattering of electrons so not coloring of proteins

• • Bulk fluorescence measurement

Freeze samples and then we could use microscopy 96 well plate which we can suck out How to dynamically switch things Plated transformed Mach I cells