Difference between revisions of "20.109(F21):M2D6"

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(Protocols)
(Protocols)
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===Part 3: Review journal article===
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===Part 2: Review journal article===
 
Read and discuss the following journal article with your laboratory partner:
 
Read and discuss the following journal article with your laboratory partner:
  

Revision as of 15:13, 30 October 2021

20.109(F21): Laboratory Fundamentals of Biological Engineering
Drawing provided by Marissa A., 20.109 student in Sp21 term.  Schematic generated using BioRender.

Fall 2021 schedule        FYI        Assignments        Homework        Class data        Communication        Accessibility

       Module 1: Genomic instability                          Module 2: Drug discovery       


Introduction

Use Kd information...

Protocols

Part 1: Complete data analysis for BLI assay

A short tutorial video on processing BLI data sets is available here (for processing) and here (for kinetic analysis). a. Open your file in the Data Analysis software. b. Save the raw file by clicking the ‘raw file’ button at the bottom. c. Align the baseline along the Y axis by clicking ‘Y-axis alignment’. d. Select the row and indicate it as ‘reference sample.’ e. We need to subtract the compound only curve from the (protein + compound) curves at during the experimental analysis. Select the rows (A1-B1) for subtraction and click the ‘subtraction’ button. f. Save the processed file by clicking the ‘process’ button. g. To obtain the kinetic binding values, we will do curve fitting to the preset binding equations. For example, there are set parameters as 1:1 vs 2:1 binding. 1:1 and 2:1 fit means 1 protein to 1 compound binding and 1 protein to 2 compound binding, respectively. We will perform both fitting operations and save the file. We will later analyse these data to see which fitting model is suitable for our kinetic experiment.


Part 2: Review journal article

Read and discuss the following journal article with your laboratory partner:

Amberg-Johnson et al. "Small molecule inhibition of apicomplexan FtsH1 disrupts plastid biogenesis in human pathogens." eLife. (2017) 6:e29865.

The initial experiment presented by Amberg-Johnson et. al. shows the effect of actinonin on apicoplast biogenesis. The apicoplast is an essential plastid organ that is a key target for drug development in research focused on malaria treatment. Actinonin was identified in large-scale screen of compounds known to inhibit growth of parasite. The subsequent experiments completed in this research served to uncover the mechanism-of-action of actinonin is it pertains to disruption of the apicoplast.

In the context of your research, this article focuses on the next step experiments that can be performed after a drug candidate is discovered from a screen. Though you can use this article as guidance as you consider the experiments that could follow your screen, remember that the specific next step experiments should be related to the protein target and drug candidate(s) identified in your project. For this exercise, the focus in on how the data are organized and presented.

In your laboratory notebook, complete the following with your partner:

  • Why is the apicoplast a promising target for anti-malarial drug development?
  • Why have attempts at developing broadly effective drugs that target the apicoplast been unsuccessful?
  • Why is the approach used by the researchers in this article more promising?
  • List the figures that are included in the article. For each figure:
    • What is the main conclusion / finding in each figure?
    • Which panel best supports the main conclusion / finding? Is more than one panel needed to fully support the main conclusion?
    • Are you convinced by the data? Do you agree with the main conclusion?
  • Are the figures organized in a coherent story?
    • Write transition statement that connect each figure to the next. A transition statement should very briefly summarize the findings of a figure and state what those findings motivated the research to do next (ie what is the next experiment?).

Reagents

Introduction

Protocols

Reagent list

Navigation links

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