Team:Harvard/Notebook
Lab Notebook
Wetlab
Got familiar with the protocols we would be using.
Model
Started to work on the mathematical model for strain engineering parameters.
Bioreactor
CAD Model and first 3D printed prototypes for BioreactorMicrofluidics
Set foundational parameters for the device.
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.
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.
Bioreactor
Created CAD model of bioreactor culture chamber and lid.
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.
Bioreactor
Created CAD model for bioreactor impeller module.
Microfluidics
Mill prototype designs on polycarbonate pieces using CNC micro-milling machine
Wetlab
We continued to repeat the pull down assay. Throughout the week, we focused on troubleshooting and minimizing performance errors.
Bioreactor
Fabricated initial 3D printing prototypes and applied waterproofing sealant.
Microfluidics
Characterized optical sensors and design circuits.
Bioreactor
Created CAD model for motor mount.
Performed water retention tests.
Microfluidics
Tested prototypes of mixing mechanisms using food coloring.
Wetlab
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
Continuous progress on the model development.
Microfluidics
Proof of concept for the microfluidic device.
Wetlab
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.
Microfluidics
- Suspended E. coli strain in pbs and measured optical density using cuvettes and nanodrop machine.
- Spun down the liquid culture at max speed for 4 minutes.
- Removed the liquid media.
- Added Pbs and diluted the culture into different concentrations.
- Vapor polished surface of the milled polycarbonate: heat up methylene chloride in a bunsen flask and place the milled area over the vapor. This was done inside a fume hood.
- Tested OD readings from vapor polished surface
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.
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.
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.
Microfluidics
Milled new designs of microfluidic device.
Microfluidics
Suspend cultures in Pbs for another round of OD calibration.
Vapor polished surface of the milled polycarbonate by heating up methylene chloride in a bunsen flask and placing the milled area over the vapor. This was done inside a fume hood.
Wetlab
We prepared parts for submission to the parts registry, as well as sequence verified our constructs.
Bioreactor
Cultured E. coli cultures in the bioreactor.
Microfluidics
Continuity of proofs of concept for the microfluidic device. Strong focus on optical sensor data analysis.
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.
Bioreactor
Cultured cells in the bioreactor, but observed minimal growth.
Microfluidics
Cultured PQN4 pBAD-CsgBACEFG (curli-producing positive control) in Kan (+) liquid media to prepare for experiments with surface treatment of microfluidic device.
Ensured OD calibration by pumping the cell cultures into microfluidic chambers via inlets of the device.
Prepared Pbs-suspended cell cuture with dilution from OD600 0.1 to OD600 1.
Used syringe pump to pump the liquid cultures in.
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.
Wetlab
Miniprepped DNA.
Bioreactor
Wetlab
We conducted PCR on each of our miniprepped parts and cloned them into the pSB1C3 backbone for sample submission according to the submission guidelines.
