(2pts) A lab technician provides you with a bacterial pellet labeled “G3”. When you observe the pellet under UV light, there is little to no fluorescence. Other than contamination, provide two scientific explanations for what might have happened to cause this outcome.
(2pts) Referring to the eight samples in Part II of the protocol, which two samples can serve as controls for background auto-fluorescence? What are the potential source(s) of auto-fluorescence in each sample？
(2pts) In the file“Exp 3 Part II data”, fluorescence data was only provided for V0. What do you predict the RFU values would have been for V1, V2, and V3? What is your basis for these predictions?
(3pts) The instructor noted increasing turbidity from G0 to G1 to G2 to G3. What is the basis for this increase? As a general rule, should sample turbidity correspond to sample fluorescence in liquid E.coli cultures? Why or Why not?
(3pts) Insert the image of the standard curve of the G0 through G3 induction data, based on you assigned data set in the file“Exp 3 Part II data”. The standard curve can be electronically constructed or hand-drawn on a graph paper (provided on the last page of the protocol). Be sure your graph has proper axes labels and a title. What was the time post-induction of the unknown sample? Mark the interpolation line on the curve (if hand-drawn) or show how you used the trend line equation (if electronically constructed) to determine the time post-induction of the unknown sample. Record your data roster number here __________________.
(2pts) A student observes 6,000 RFUs in a 200 μl aliquot from a G3-500 ml culture (a 500 ml culture of G strain, induced for 3 hours). How many total RFUs will be present in a G3-15ml sample? During the rGFP purification in lab#4, assume the student recovers 75% of the fluorescing protein from the G3-15ml sample. What would be the maximum total number of RFUs that the student collected in the purification procedure? Show your work (either typed, as an inserted image, or annotated in the document) for full credit.
(10 pts) Using the fluorescently-tagged protein localization images presented in Part III of the protocol, match the image (by number) with the experiment description/results below and explain your reasoning.
Histone H2B-GFP and Ras-RFP were visualized during the earliest stages of embryonic development.
Image # ________
Reasoning:
The extracellular domain of a growth factor receptor was tagged with GFP and its ligand was labeled with RFP.
Image # ________
Reasoning:
Comet-RFP and Temoc-BFP were both found to be distributed throughout the cytoplasm of cultured mouse cells.
Image # ________
Reasoning:
Microtubule-associated protein-GFP and histone H3-RFP were visualized in cells at high magnification.
Image # ________
Reasoning:
Protein E01-RFP and S88-GFP are expressed and co-localize within vesicles or organelles in a subset of cells in the tissue analyzed.
Image # ________
Reasoning:
(4pts) Based on the promoter assay data provided in Part III of the protocol, how do compounds U, T, and D, affect the Whoosh promoter? Predict the impact of each compound on the transcription of Whoosh protein.