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20.109(F20): Laboratory Fundamentals of Biological Engineering

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       M1: Genomic instability        M2: Drug discovery        M3: Metabolic engineering       


Schematic of RNAP, σ factor, promoter complex. A. The core RNAP enzyme is composed of five subunits: β, β', two α units, and ω. When the core RNAP enzyme is associated with a σ factor, the holoenzyme is formed. B. The holoenzyme binds to DNA promoters at the -10 / -35 sites that are specific to the σ factor. C. When the holoenzyme is associated with DNA it is in an open complex formation that enables transcription.
In the initial experimental approach used to increase ethanol yield using the CRISPRi system, the previous 109 students considered only a simplistic model for gene expression when designing sgRNA_target sequences: transcription initiation occurs when RNAP binds to the promoter regions and elongation proceeds as RNAP reads the DNA sequence to generates transcript. To inhibit expression, sgRNA_target sequences were designed to block one of these processes using the dCas9 protein as a roadblock. In actuality, regulation of gene expression in bacteria is far more complex.

The core RNAP enzyme is composed of five subunits: β, β', αI, αII, and ω. The β and β' subunits form a 'claw' structure that are joined together by the α-dimer. The N-terminal domains of the α-dimer are in contact with the β and β' subunits and the C-terminal domains contain a flexible linker that is able to associate with DNA and / or regulatory proteins. The ω subunit is involved in assembly of RNAP. When this core enzyme is complexed with a σ factor, the holoenzyme is formed. The σ factor is critical in RNAP binding to DNA as it binds to the -10 / -35 sites within the promoter. In bacterial cells σ are classified into two distinct families, with several species also encoding alternate σ factors that regulate specific sub-sets of genes in specific conditions. In addition to σ factors, additional regulators are important in regulating gene expression in response to environmental cues.


Part 1: Examine targeted gene sequences for regulatory binding sites

Today you will examine the DNA sequences of the targeted genes to identify features that may impact sgRNA_target binding. To do this you will use an online tool that predicts transcription factor binding sites (TFBS) in prokaryotic DNA sequences. Because you will be collecting a lot of information today for ldhA and pta-ack, it is important that you keep clearly organized notes in your laboratory notebook. As in the previous exercises, feel free to divide the workload between laboratory partners.

  1. Use the KEGG Database to obtain the DNA sequences of the targeted genes (ldhA and pta-ack) in the E. coli K-12 MG1655 strain.
    • Enter the name of targeted gene in the Search genes box and click Go.
    • Because sgRNA_target molecules were generated that target the promoter, enter 50 in the +upstream box to get the 50 basepair sequence immediately preceding the start codon.
  2. Copy the the sequence for the targeted gene and paste it into the Virtural Footprint interface.
    • In the section labeled 'Step 1: Input DNA Sequence...', insert the sequence from KEGG into the 'Paste Sequence' box.
  3. Next, in the section labeled 'Step 2: Select Pattern and Start Searching...' for the Position Weight Matrix click the 'Select All' button.
  4. Then click the 'Start' button.

Part 2: Discuss your Research proposal with peers

To help you focus your ideas and develop the details of your project, you will discuss the project description you submitted today with a classmate from another group. As you listen to your classmate's idea, consider the following criteria proposed for small research project grants by the NIH:

Small Research Grant Program: ...small grant supports discrete, well-defined projects that realistically can be completed in two years and that require limited levels of funding. Because the research project usually is limited, the grant application may not contain extensive detail or discussion. Accordingly, reviewers should evaluate the conceptual framework and general approach to the problem. Appropriate justification for the proposed work can be provided through literature citations, data from other sources, or from investigator-generated data. Preliminary data are not required, particularly in application proposing pilot or feasibility studies.

Use the following exercise to guide your discussion as you consider both your and your classmate's project. Because you are still in the early stages of developing your research topic, it is okay if you do not have all of the answers to the following questions. This is meant to help you critically think about your proposal...not to point out the additional research you need to complete! Furthermore, this is an informal conversation and you should feel free to look up information during this exercise or just make notes so you know what to research later with your co-investigator.

Outline of the peer review exercise:

  • Find the partner you were assigned by the teaching faculty and begin by deciding which partner will present first.
    • As the presenter, focus on why you believe your topic is important and provide the context needed to convince your listener that it is indeed worth pursuing.
    • As the listener, verbally summarize the topic back to the presenter to ensure you understood the proposal. Are you convinced that the topic is important? Why or why not? Discuss this with the presenter.
  • Now that you have the needed background information discuss the 'Hows' of the project.
    • As the presenter, consider the questions below as you give some details about your proposal.
    • As the listener, feel free to ask questions and maybe provide some helpful feedback as the presenter discusses the details of their project.

Questions to guide your discussion:

  1. What is the novel aspect of your proposal?
  2. Why do you believe your project is feasible?
  3. Is there evidence that supports your proposal?
  4. How will your research advance the field?
  5. How will you complete your research (what methods might you use)?
  6. What is the expected result?
  7. How might you 'double-check' or confirm an expected result?
  8. What if you do not get the expected result?
  9. What can be learned if you get the expected result? If you get an unexpected result?
  10. What are some alternative approaches (methods) for your proposed research?

Once you have completed discussing the presenter's project, switch roles and complete this exercise with the listener's project.

In your laboratory notebook, complete the following:

  • Record the notes from your discussions with your peers.
    • What insights regarding your own project were provided by your peers?
    • What did you find interesting about your peers projects?

Part 3: Consider societal implications of your Research proposal

Thus far we have focused on the experiments that you are designing as part of the Research proposal presentation; however, another important aspect of this assignment is defining the societal impacts of your proposed work. In recent years, funding agencies have placed increased emphasis on societal impact when reviewing grant applications and the public has been more critical of the use of government funds toward research that does not benefit the population. This sentiment is expressed in a recently published editorial:

"...research funding agencies will no longer be satisfied with claims that our research has impact merely because we use it in training of our students, because it is well-cited by other academics, or because it is published in reputable journals...it seems reasonable that at least some discernible societal value should emerge from research." (Davison and Bjorn-Anderson. Info Systems J. 2019;29:989-993)

Read and discuss the following perspective with your co-investigator:

Frodeman and Holbrook. "Science's social effects." Issues in Science and Technology. Vol. XXIII, No. 3, Spring 2007.

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

  • What are the societal implications of your proposed research?
    • Which populations benefit from your research? Is it possible any populations will be negatively impacted by your research?
    • Will cost limitations impede / bias which populations are benefited?
    • Is your research applicable to all populations?
  • What are the ethical implications of your proposed research?
    • Will you use harmful substances in your experiments?
    • Will you generate potentially harmful materials in your research?
    • Will you collect personal information as part of your research?
  • If sampling / testing the population, how will you select the subject pool?
    • Is equal representation across gender / race / sex / ethnicity important? Why or why not?
  • If using animals, how will you determine if experiments are necessary?
    • How will you ensure humane treatment of animals during experiments?

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