20.109(F13): Mod 2 Day 2 Tools for Systems Biology

Introduction

How does one define 'Systems Biology'? One definition is provided by the MIT Integrative Cancer Biology Program:

Systems biology strives to describe the extreme multivariate nature of cellular systems using statistical and mathematical techniques, ultimately predicting the response of cells/tissues/organisms to normal and pathological perturbations. To achieve predictive models, systems biology demands integration of disparate data; genomic, proteomic, metabolomic, or epigenetic data alone is not enough to fully describe organismal behaviors. Therefore, systems biology is necessarily a multidisciplinary venture requiring significant collaboration and melding of experimentation and computation.

Phew, that is a mouth full. Let's boil that down to two key points: Systems biology depends on (1) measuring many parameters and (2) using mathematical relationships to distill those parameters down to the ones that are most important. To satisfy these key points we need an informative experimental system that is amenable to a great number of perturbations. For the purpose of 20.109, 'perturbation' refers to the cell stimulus or intracellular signaling pathway inhibitor that we are employing in this module. However, in a greater context, perturbation could be cell culture media formulation, temperature and oxygen conditions within the TC incubator, elasticity of cell culture surface, 2D vs. 3D culture environment, etc.

Figure 1 from Hamden et al., Current Protocols in Neuroscience Unit 5.23 (2006)

Today we will ready the components for our systems biology study. To measure activation of the EGFR signaling network using BRET, we need to introduce a donor protein and an acceptor protein into the cell. In our experiment, the donor protein is a SH2 domain fused to renilla luciferase (RLuc). In Part 1 of today's lab, you will transform your RLuc-SH2 domain plasmid into DH5a' competent bacteria following a protocol that is similar to the one you completed in Module 1. We need to grow a lot of plasmid to do our HTS study and you will do the grunt work for your own experiment here!

As illustrated in Figure 1, we also need an acceptor fluorescent protein (FP in the figure). In our case we are using enhanced GFP (or EGFP) tagged to the C-terminus of EGFR. Borrowing cells from the work of Harms et al., we will employ a chinese hamster ovary (CHO) cell line that has approximately 600,000 EGFR-EGFP proteins on the cell surface. As you may remember, EGFR signaling drives cell proliferation and migration in cancer and the range of EGFR found in carcinoma cells is XX to XX, making our CHO cell model ideal for simulating EGFR network activation in tumors. CHO cells also lack endogenous EGFR, so we can be confident that all of the effects of EGF are mediated through our engineered EGFR-EGP. Take a minute and think about why that might be important when creating a system to study EGFR signaling.

Lab time will be split into two parts today, half the class will start in tissue culture (Part 1) and half will start in the main lab (Part 2). The red, yellow, blue, purple, and platinum groups will start on Part 1 and the orange, green, pink, and white groups will start on Part 2.

Part 1: Bacterial Transformations

You will perform 3 bacterial transformations today using DH5a' cells in order to grow enough of your RLuc-SH2 domain and a negative control RLuc-only plasmid for the culminating HTS on M2D7. Why are there three reactions if we only have two plasmids to prep?

1. Prewarm and dry three LB+AMP plates by placing them in the 37°C incubator, media side up with the lids ajar.
2. Label three eppendorf tubes with your transformation products and place on ice to pre-chill them.
3. Get an aliquot of competent cells from one of the teaching faculty. Keep these cells on ice at all times. There should be at least 150 μl of cells in each tube. Aliquot 50 μl of cells into 3 clean eppendorf tubes.
4. Add 1 μl DNA or 1 μl sterile water to each tube of cells.
5. Flick to mix the contents and leave the tubes on ice for 10 minutes.
6. Heat shock the cells at 42°C for 90 seconds exactly and put on ice for two minutes. Use your timer.
7. Move the samples to a rack on your bench then use your P1000 to add 0.5 ml of LB media to each eppendorf tube. Invert each tube to mix.
8. Move the tube rack to the 37°C incubator for 30 min to allow bacteria to begin proliferating.
9. Plate 100 μl of each transformation mix on LB+AMP plates.
   Note: After dipping the glass spreader in the ethanol jar, then pass it through the flame of the alcohol burner just long enough to ignite the ethanol. After letting the ethanol burn off, the spreader may still be very hot, and it is advisable to tap it gently on a portion of the agar plate without cells in order to equilibrate it with the agar (if it sizzles, it's way too hot). Once the plates are done, wrap them with colored tape and incubate them in the 37°C incubator overnight. One of the teaching faculty will remove them from the incubator and set up liquid cultures for you to use next time.

Notes for Teaching Faculty

TA notes, mod 2