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5.3.1: Module 1 Assignment- RNA Engineering Laboratory Report - Biology

5.3.1: Module 1 Assignment- RNA Engineering Laboratory Report - Biology



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Overview

The culminating assignment for Module 1 will be a laboratory report in which you describe your RNA engineering investigation. It is essential that you relate not merely what you did but why you did it, and not only what your data presently shows but what it means for the future. Thus, you can assume rapid comprehension – but not a priori knowledge – of technical information, and consequently should strive to present your work in a logical, step-by-step fashion.

Be sure to review the 20.109 statement on collaboration and integrity.

Logistics

First Draft Submission

The first draft of your research article is due by 11 am on Day 1 of Module 2.

Revised Article Submission

Your first draft, with feedback from both the writing and the technical faculty, will be returned on Day 4 of Module 2 (9 days later). You will then have the opportunity to revise your report for up to a one and one-third letter grade improvement. In other words, a C can be revised up to an B+, a C+ to an A-, a B- to an A, etc.) The final draft is due one week later, on Day 6 of Module 4. Please highlight any substantial revisions to your text, for example, by using a different colored font.

Formatting Expectations

  • Your main document (excluding figures) should be/have
    • .doc (preferred) or .pdf
    • 12-pt font
    • with 1-inch margins
    • double-spaced (excepting the abstract)
  • Figures can be made in a separate drawing program (such as powerpoint), and should be submitted as .pdf

Guidelines on Length

Not counting figures, report length should be about 10-13 pages, and certainly not exceed 15 pages.

Though somewhat variable, typical section lengths might be:

  • Introduction: 2-3 pages
  • Methods: ~3 pages
  • Results: 3-4 pages
  • Discussion: 3-4 pages

Content Guidelines

Begin by reading the general guidelines for writing up your research, which describe the expectations for every section of the report, from Abstract to References. A few notes specific to Module 1 are as follows.

Introduction

You are welcome to use your own creativity and judgement as to what a good introduction should look like; however, you may find the suggested structure (see also general guidelines) and content below useful. One approach you may choose is to emphasize method optimization to motivate your introduction, and to address the following guiding questions:

  • Paragraph 1
    • What is SELEX?
    • What are some benchmarks (in your own reasoning) that if attained, would maximize SELEX’s efficiency and accessibility.
    • What are some key parameters to optimize in achieving your desired efficiency?
  • Paragraph 2
    • Which parameter(s) have you chosen to investigate in your present study?
    • What is the rationale for choosing to explore this parameter in the context of improving selection efficiency? (That is, how is this parameter linked to selection efficiency?)
  • Paragraph 3
    • Why did you choose your specific conditions? [Consider this in the context of the parameter space covered as a lab section, in addition to what you are doing individually]
    • Your expectations for how the outcome will vary as a function of your explored parameter space.
    • A brief summary of how you intend to assess whether your experiment worked (yours individually, and pooled across your lab section).
    • A brief overview of your results and conclusions.

Suggested Figures

Your report is expected to contain more or less the following figures. Of course you are welcome to make modifications and additions as you see fit. Recall that figures should generally be described in the Results section.

  • Schematics/diagrams
    • Schematic showing overall experimental plan and main steps involved
  • Figures
    • Gel from initial PCR
    • Gel with RT-PCR samples
    • Binding curves for your own set of data
  • Tables or just text
    • Binding data (peak height and peak shift) for entire lab section (or entire class if available in time)

References

You are not expected to do a thorough survey of the relevant primary literature for this first report. However, your introduction (and potentially discussion) should contain a total of at least three references.

Evaluation

The full descriptive rubric for lab reports can be found on the guidelines for writing up your research. The weighting for Module 1 is as follows:

SECTIONWORTH (%)
Title2
Abstract5
Introduction20
Materials and Methods15
Results25
Discussion20
References3
Writing10

5.3.1: Module 1 Assignment- RNA Engineering Laboratory Report - Biology

Lab work is divided into three modules of eight sessions each. Learning materials for each lab session (linked below under 'Lab Materials') include an introduction, experimental protocol, and a reagents list, followed by a "for next time" homework assignment. A short pre-lab lecture preceeds most of the lab sessions.

Selected results from some labs are included courtesy of the students and used with permission.


Abstract

To overcome environmental problems caused by the use of fossil resources, microbial cell factories have become a promising technique for the sustainable and eco-friendly development of valuable products from renewable resources. Constructing microbial cell factories with high titers, yields, and productivity requires a balance between growth and production to this end, tuning gene expression and regulation is necessary to optimise and precisely control complicated metabolic fluxes. In this article, we review the current trends and advances in tuning gene expression and regulation and consider their engineering at each of the three stages of gene regulation: genomic, mRNA, and protein. In particular, the technological approaches utilised in a diverse range of genetic-engineering-based tools for the construction of microbial cell factories are reviewed and representative applications of these strategies are presented. Finally, the prospects for strategies and systems for tuning gene expression and regulation are discussed.


Daily Work

In addition to the assignments listed above there will be

  • Daily Lab Quizzes (5% of final grade)
    • These are intended to refresh your memory about the experiment you are performing. They will not be hard and should take just 5-10 minutes at the beginning of lab.
    • You will record your data on the white pages of a bound notebook. The yellow, duplicate pages will be collected and evaluated by the teaching assistants.
    • Notebooks will be evaluated according to the criteria described here.
    • These will vary considerably in content and associated points/weighting. Most of them will directly prepare you for major assessments (e.g., making a draft figure for a lab report) or lab work (e.g., performing a calculation in advance).
    • The homeworks can be found in the "for next time" (FNT) section of each lab day as well as in a single list here.
    • Assignments should be submitted as hardcopies at the beginning of lab. A select few assignments must also be submitted on Stellar in order to receive feedback from the writing instructors and are marked as such.
    • You can work with your lab partner, friends and teaching assistants on the FNTs but you will hand in individual assignments unless otherwise specified.
    • As a student in 20.109, you are expected to be an active participant in a scientific community. Your student colleagues, the teaching faculty, and especially your lab partner are all your collaborators. They rely on you for timely posting of your data, and for your unique and thoughtful contributions during class.
    • One-third of your participation grade will be directly assigned by the teaching faculty, who will consider: whether you asked and/or answered questions during lecture, whether you engaged with opportunities to improve your understanding/communication/etc., and whether you promoted a considerate and collaborative class environment.
    • To determine the other two-thirds of your participation grade, you will write brief reflective pieces on a few aspects of your 20.109 experience throughout the semester. These assignments will be listed under that day's FNT section, but will be counted separately from other homework. You must complete at least 4 and no more than 8 reflections. The due date schedule and further explanation of both mandatory and optional reflections is linked here.

    5. Tackling sizeable problems in RNA biophysical chemistry

    5.1 Structural basis for encapsidation of the genome of the Moloney Murine Leukemia Virus

    100 nt can direct RNA packaging into virus-like particles, albeit at reduced efficiency. To gain insight into the molecular basis of genome packaging in MMLV, D'Souza and Summers determined, using NMR, the 3D structure of the 101 nt RNA sub-fragment of the Ψ-site in complex with a nucleocapsid domain of the Gag polyproteins important for mediating this interaction [107]. To tackle the largest known NMR RNA structure, the authors had to use a nucleotide-specific isotopic labeling strategy to overcome the extensive chemical shift overlap [61]. These methods are analogous to those described in making labeled NTPs in sections 3.0–3.4 above. With this approach, they were able to distinguish internucleotide from intranucleotide nuclear Overhauser effects (NOE), and obtain valuable distance information to compute a moderate resolution structure [61, 107]. The disadvantage of this approach is that it required preparing eight different isotopically labeled samples and collecting several 2D, 3D, and 4D NMR datasets, making it both labor and instrumentation-intensive [61]. Uses of some of the technologies discussed above are likely to help with getting higher resolution structures of this biologically important complex.


    20.109(S12):Module 1

    In this module you will investigate RNA aptamer selection. You may already be familiar with biological entities that bind to specific molecules, such as antibody proteins or their peptide derivatives. Short fragments of RNA can also have high-order structures that allow them to bind a target molecule with good affinity and specificity successful binders are called aptamers. Normally, RNA aptamers that bind particular targets are found by screening many candidates at random in a process called SELEX, or systematic evolution of ligands by exponential enrichment. (Predictive computational tools can also be used to design rather than select aptamers.) In the coming weeks, you will essentially perform one round of SELEX. Because SELEX typically takes several rounds to isolate target-binding aptamers, you will start with a known RNA mixture rather than than a completely random library. Your goal will be to explore what experimental parameters affect the enrichment of a heme-binding RNA aptamer from a mixture of heme-binding and non-binding RNAs.

    We thank 20.109 instructor Natalie Kuldell for helpful discussions and for acquiring funding for module development.


    Watch the video: Week #1 Lab Report #1 (August 2022).