20.109(S14): Choose system conditions and paper discussion (Day3)

Introduction

Today you will become intimately familiar with the plasmid reporter assay for measuring NHEJ that we will use in Module 2. You will examine the different elements present in the plasmid, and more importantly you will try to reconstruct some of the design choices that were made when different variants of the reporter were conceived. We will also continue learning about Ku80, this time through a class-wide discussion of a recent research article on the role of Ku80 in lung cancer.

A key topic that you will need to understand to complete today’s (re-)design exercise is the function and (sequence-level) structure of restriction enzymes.

topic 1: restriction enzymes

topic 2: design considerations in a repair reporter assay

Protocols

Part 1: Paper discussion

Technical Background

The main technical topic that may be unfamiliar to (some of) you is the use of short interfering RNA (siRNA). RNA interference, or RNAi, is a post-transcriptional silencing mechanism. You may have heard of gene "knock-outs" before, and in fact xrs6 are essentially Ku80 knock-outs: the DNA sequence is deleted or modified so as to be non-functional. In contrast, RNAi is usually not as potent, and hence is called gene "knock-down." Here the DNA sequence is normal, and mRNA is transcribed – but it is bound up or destroyed before it can be translated. You can read more about the intricacies of RNAi at Scitable.

Discussion Topics

Writing

• How does this abstract style differ from the one you have previously encountered – and also explicitly been taught – in 20.109? How is it the same? What are the pros and cons of each format? As a whole, did the abstract make you want to read the paper?
• This Introduction is short but packs a punch. With the 20.109 guidelines in mind, locate the key elements of an introductory section here. What one sentence best defines the research gap?
• Let's pay special attention to the Methods section, since you will be writing one during Module 2!
  • What best practices for Methods section writing do the authors follow? For example, do they write clear topic sentences of appropriate scope?
  • What is the authors' strategy for sub-section groupings? What is the role of the first sub-section?
  • What methods do the authors seem to assume that most readers will be familiar with?
  • What methods do the authors seem to assume need more detailed definition or citation?
  • What is the purpose of a passage beginning "Briefly, …"?
  • Are there any changes you would suggest the authors make?
• We'll talk about the writing style in the Results and Discussion sections in tandem with our conversation about the technical content. Remember to keep in mind
  • Results guidelines,
  • Discussion guidelines, and
  • differences between the two.

Content

The following questions will guide our in-class discussion; consider them as a starting point rather than a check-list.

• Figure 1 (Note: caption mis-states which letter label corresponds to which image)
  • Describe the primary finding(s) in this Figure and associated Results text.
  • What do you think the purpose is of showing both qualitative and quantitative data?
  • Would you have expected, a priori, for A and B to look identical? Why or why not?
  • How do you interpret the y axes in figures C and D?
• Figure 2
  • Describe the primary finding(s) in this Figure and associated Results text.
  • Looking back at the Methods, what did the authors do to obtain as credible data as possible?
• Table 1
  • Describe the primary finding(s) in this Table and associated Results text.
  • How do these findings relate to the central question of the study?
• Figure 3
  • Describe the primary finding(s) in this Figure and associated Results text.
  • What is the unit on the x axes?
  • Looking back at the methods, define the survival parameters, and explain how the authors dealt with potentially confounding data.
• Table 2
  • Describe the primary finding(s) in this Table and associated Results text.
  • Would you want to modify this Table to include the information in the main body text? If so, how?
• Figure 4
  • Describe the primary finding(s) in this Figure and associated Results text.
  • What is the role of the lower strip in each of A and B? How is this information used in panels C and D?
  • What is the purpose of switching from investigating primary tumors to investigating a cell line?
  • Relatedly, what is the purpose of obtaining the data for Figure 4 before moving on to the experiments in Figure 5?
• Figure 5
  • Describe the primary finding(s) in this Figure and associated Results text. How do the purposes of the different panels differ? Which ones might you group together?
  • What is the meaning and the role of the "scramble" siRNA?
  • There is a one-word mistake in the associated text – where, and what should the word be instead?
  • Looking back at the Methods, how do you interpret the different quadrants of the flow cytometry data plot?
• Discussion
  • What new information do we learn here? What types of information tend to come up?

Part 2: Reverse engineering pMax-BFP-MCS

Coming later!

1. Use Lonza website and pMax-BFP-ScaI file to identify general plasmid features and understand Zac's original design. 2. Put sequence in NEBcutter to assess potential restriction enzyme choices. How would you narrow down? 3. Use pMax-BFP-MCS file to understand 20.109 design.

1. Begin by downloading the pMax-BFP-ScaI file. This plasmid is an NHEJ assay developed in Samson lab...

Part 3: Choose system conditions

On today's Talk page...

For next time

digest planning and calculations in advance

Reagent list

Just your brains!

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