Wetlab

In order to screen the best assay for our intended project, we tried multiple protocols to measure the quantity of produced curli, the desired polymer to optimize the production of.

Growing curli producing E. coli on plates with congo red:*


Isolating curli by vacuum filtration:*


Running a congo red pull down assay:*


From running the protocols in parallel we decided that the pull down assay served as the most consistent proxy for curli production. It was difficult to quantify the "redness" of the cultures on the red plates, and we did not notice significant difference between controls. For the vacuum filtration, it required large volumes of culture and small quantities of the yield product.

*Detailed procedure found on our protocol page.

Wetlab

To ensure consistency of results, we ran through multiple iterations of congo red pull down assays with multiple aliquots of the same liquid culture. In the beginning we had different absorbance readings for the same culture, suggesting procedural error. Throughout the week, we focused on troubleshooting and minimizing performance errors.

Interlab

Data was collected for the interlab study.

Wetlab

To ensure consistency of results, we ran through multiple iterations of congo red pull down assays with multiple aliquots of the same liquid culture. In the beginning we had different absorbance readings for the same culture, suggesting procedural error. Throughout the week, we focused on troubleshooting and minimizing performance errors.

Interlab

Data was collected for the interlab study.

Wetlab

To ensure consistency of results, we ran through multiple iterations of congo red pull down assays with multiple aliquots of the same liquid culture. In the beginning we had different absorbance readings for the same culture, suggesting procedural error. Throughout the week, we focused on troubleshooting and minimizing performance errors.

Interlab

Data was collected for the interlab study.

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.