Difference between revisions of "Team:Harvard/Notebook"
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<h2>Wetlab</h2><br> | <h2>Wetlab</h2><br> | ||
| − | 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 | + | 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. | |
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<br> | <br> | ||
<h2>Interlab</h2><br> | <h2>Interlab</h2><br> | ||
Data was collected for the <a href="https://2017.igem.org/Team:Harvard/InterLab">interlab study</a>. | Data was collected for the <a href="https://2017.igem.org/Team:Harvard/InterLab">interlab study</a>. | ||
| + | <br> | ||
| + | <h2>Model</h2><br> | ||
| + | Started to develop the mathematical model to see which gene we wanted to edit in the wild type strain of <i>E. coli</i>. This involved reading many papers on measurements of relevant parameters, such as secretion rate, rates of translation, transcription, etc. | ||
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| − | <div class="panel-body"> | + | <div class="panel-body"> |
| + | <h2>Wetlab</h2> | ||
| + | We continued to repeat the pull down assay. Throughout the week, we focused on troubleshooting and minimizing performance errors. | ||
| + | <br><br> | ||
| + | <center> | ||
| + | <b>Inconsistencies among triplicates</b><br> | ||
| + | <img src="https://static.igem.org/mediawiki/2017/1/1d/Harvard--variable.png"> | ||
| + | </center> | ||
| + | <br><br> | ||
| + | <center> | ||
| + | <b>Consistent samples at the end of the week</b><br> | ||
| + | <img src="https://static.igem.org/mediawiki/2017/9/95/Harvard--Clear.jpeg"> | ||
| + | </center> | ||
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Revision as of 22:52, 1 November 2017
Lab Notebook
Created the mathematical model for strain engineering parameters.
Started basic research about microfluidic device and bioreactor framework.
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.
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.
Panel Body
Panel Body
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.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.
Panel Body
Wetlab
We prepared parts for submission to the parts registry, as well as sequence verified our constructs.
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. collected data for the InterLab study and ran the congo red pulldown assay again.
We ran the congo red assay for a third time.
Panel Body
Wetlab
We conducted PCR on each of our miniprepped parts and cloned them into the pSB1C3 backbone for sample submission.
