20.109(F16):Induce CRISPRi system (Day7)

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20.109(F16): Laboratory Fundamentals of Biological Engineering

Engelward PNAS 2006.png

Schedule Fall 2016        Announcements        Assignments        Homework        Communication
       1. Measuring Genomic Instability        2. Manipulating Metabolism        3. Engineering Biomaterials              

Introduction

dual plasmid system...

aTc induction...

Protocols

Part 1: Communication Lab workshop

Our communication instructors, Dr. Sean Clarke and Dr. Diana Chien, will join us today for a workshop on organizing and structuring your Research article.

Part 2: Examine psgRNA sequencing results

Your goal today is to analyze the sequencing data for you two potential mutant IPC samples - two independent colonies from your X#Z mutant - and then decide which colony to proceed with for the X#Z mutant.

Retrieve sequence results from Genewiz

  1. Use the pRSET-IPC ApE file to mark and/or note down the expected location of your mutation before proceeding.
    • You can simply compare to your annotation of the IPC alone ApE file that you prepared on Day 1 of the module.
    • You may also find it helpful to generate another ApE file with only the CaM portion of IPC and use this when you assess the Genewiz sequencing results.
  2. Your sequencing data from Genewiz is available at this link.
    • Choose the "Login" link and then use "nllyell@mit.edu" and "be20109" to access your results.
    • At the bottom right should be a link to download your sequencing results.
      • TR section: click on the Tracking Number 10-324382914 (Order Date 02-18-2016) and Tracking Number 10-324581564 (Order Date 02/21/2016)
      • WF section: click on the Tracking Number 10-324426168 (Order Date 02-19-2016) and Tracking Number 10-324584038 (Order Date 02/21/2016)
  3. The quickest way to start working with your data is to follow the "View" link under the Seq File heading. For ambiguous data, you may want to look directly at the Trace File as well.

You can align your sequencing data with a known sequence, in this case the CaM portion of inverse pericam, and the differences will be quickly identified. There are several web-based programs for aligning sequences and still more programs that can be purchased. The steps for using APE and the NCBI-hosted tool are below. Please feel free to use either program...or any program with which you are familiar.

Align with Benchling

If both colonies for your mutant have the correct sequence, choose one to use for the protein purification step. If only one is correct, then this is the one you will use next time. If neither of your plasmids carry the appropriate mutation, talk to the teaching faculty.

Part 3: Prepare media for mixed-acid fermentation inoculations

Reagents

Navigation links

Next day: | Measure fermentation products

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