Wetlab

Got familiar with the protocols we would be using.

Model

Started to work on the mathematical model for strain engineering parameters.

Microfluidics and Bioreactor

Started basic research about microfluidic device and bioreactor framework.

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 liquid cultures, suggesting procedural error.

Interlab

Data was collected for the interlab study.

Model

Started to develop the mathematical model to see which gene we wanted to edit in the wild type strain of E. coli. This involved reading many papers on measurements of relevant parameters, such as secretion rate, rates of translation, transcription, etc.

Wetlab

We continued to repeat the pull down assay. Throughout the week, we focused on troubleshooting and minimizing performance errors.

Inconsistencies among triplicates


Consistent samples at the end of the week

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Wetlab

We focused on generating the RBS library for strain engineering. Once we received the DNA parts, we used selection markers to appropriately transform, clone, and sequence the optimized strain.

Model

There was continuous progress on the model development.

Wetlab

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.

Wetlab

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.
PCR banding patterns indicated successful Gibson Assembly

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. Example of imaged plates: Brightest colonies related to higher curli production

Wetlab

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.

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Wetlab

We prepared parts for submission to the parts registry, as well as sequence verified our constructs.

Wetlab

We ran the congo red pulldown assay again for our constructs.
We sequenced our constructs, and analyzed the sequence in context of our model and other parameters. Refer to our results for a detailed analysis.

Interlab

We prepared our interlab data for submission. We also collected additional data for the interlab study and ran the congo red pulldown assay again.

Wetlab

We ran the congo red assay for a third time. We also sent more of our constructs to be sequenced since not all of the parts were able to be sequenced the first time.
Final data refer to href="https://2017.igem.org/Team:Harvard/Results" results for more details

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Wetlab

We conducted PCR on each of our miniprepped parts and cloned them into the pSB1C3 backbone for sample submission.