Team:Harvard/Notebook
Lab Notebook
We designed an RBS library for csgG using the Salis Lab RBS Library Calculator, as well as the appropriate PCR primers to construct the library and ordered these sequences from IDT.
Day 1
Our DNA parts ordered the previous week arrived and we conducted PCRs with the primers we designed and a plasmid containing the sequences for csgA, csgB, csgC, csgE, csgF, and csgG obtained from the Joshi Lab to modify the RBS sequence in front of csgG.Day 2
After verifying our PCR products with a gel, we used Gibson Assembly to put our cloned parts in an expression vector with kanamycin resistance. We then transformed our newly formed plasmids into competent cells using heat shock and plated them on agar plates with kanamycin.Day 3
After leaving our plates in an incubator overnight, we imaged the plates FluorChem E and ran an image analysis script on the images to determine the brightest colonies, which correspond to the colonies with highest curli production. We then picked the 2 brightest colonies on each plate, as well as 2 other randomly chosen colonies, and cultured them in 5 mL falcon tubes with liquid LB and kanamycin.We ran a congo red pulldown assay on the cultures from the previous week to quantitatively measure the amount of curli produced. Then, we miniprepped the cell cultures to send out our parts for sequencing.
No lab work :(
We collected data for the InterLab study and ran the congo red pulldown assay again.
We ran the congo red assay for a third time.
No lab work :(
We conducted PCR on each of our miniprepped parts and cloned them into the pSB1C3 backbone for sample submission.
