Difference between revisions of "Team:Harvard/Notebook"

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Created the <a href="https://2017.igem.org/Team:Harvard/Model">mathematical model</a> for strain engineering parameters.
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        <br>Started basic research about <a href="https://2017.igem.org/Team:Harvard/Microfluidic">microfluidic device</a> and <a href="https://2017.igem.org/Team:Harvard/Bioreactor">bioreactor</a> framework.
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<h2>Wetlab</h2><br>
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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. <br><br>
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<center>
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<b>Growing curli producing <i>E. coli</i> on plates with congo red:*</b><br>
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<img src ="https://static.igem.org/mediawiki/2017/c/c8/Harvard--CR.png"></center> <br><br>
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<center><b>Isolating curli by vacuum filtration:*</b> <br>
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<img src="https://static.igem.org/mediawiki/2017/e/ef/Harvard--Vacuum.png"></center><br><br>
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<center><b>Running a congo red pull down assay:*</b><br>
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<img src="https://static.igem.org/mediawiki/2017/4/41/Harvard--Pulldown.png">
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</center>
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<br><br>
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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.
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<br><br>
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*Detailed procedure found on our <a href="https://2017.igem.org/Team:Harvard/Protocols">protocol page</a>.
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<h2>Wetlab</h2><br>
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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.
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<br><br>
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<h2>Interlab</h2>
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Data was collected for the <a href="https://2017.igem.org/Team:Harvard/InterLab">interlab study</a>.
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We designed an RBS library for csgG using the <a href="https://salislab.net/software/">Salis Lab RBS Library Calculator,</a>as well as the appropriate PCR primers to construct the library and ordered these sequences from IDT.
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<h2>Wetlab</h2>
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<h5>Day 1</h5>
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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.
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<h5>Day 2</h5>
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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.
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<br><br>
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<h5>Day 3</h5>
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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.
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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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We collected data for the InterLab study and ran the congo red pulldown assay again.
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We ran the congo red assay for a third time.
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We conducted PCR on each of our miniprepped parts and cloned them into the pSB1C3 backbone for sample submission.
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Revision as of 22:22, 1 November 2017

Harvard OPTI POLY

Lab Notebook


Month 1: August Overview

Created the mathematical model for strain engineering parameters.
Started basic research about microfluidic device and bioreactor framework.

Week 1: 8/7 - 8/12

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.

Week 2: 8/14 - 8/18

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.

Week 3: 8/21 - 8/25

Panel Body

Week 4: 8/28 - 9/1

Panel Body


Month 2: September Overview

Panel Body

Week 5: 9/4 - 9/8

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.

Week 6: 9/11 - 9/15

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.

Week 7: 9/18 - 9/22

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.

Week 8: 9/25 - 9/29

Panel Body


Month 3 October Overview

Panel Body

Week 9: 10/2 - 10/6

We collected data for the InterLab study and ran the congo red pulldown assay again.

Week 10: 10/9 - 10/13

We ran the congo red assay for a third time.

Week 11: 10/16 - 10/20

Panel Body

Week 12: 10/23 - 10/27

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