Question

Answer the following questions that do not have an answer. Creating a graph is not necessary. Thanks!!!

You introduced the pGlo vector into E. coli which carried the GFP gene under the control of the arabinose promoter.  Based on what you know about the arabinose promoter, when did you expect GFP to be expressed?

I expected the green fluorescence protein to be expressed when arabinose was present.

What hypothesis was tested by the experiments you performed after the pGlo vector was introduced into E. coli?

We tested one hypothesis: Will other sugars other than arabinose, such as glucose, lactose & fructose, trigger the arabinose promoter?

Which techniques were used and what did each one measure?

Two techniques were used: Fluorescence using spectrophotometry and qRT-PCR. Spectrophotometry was used to measure the fluorescence in the cultures exposed to different sugars. If the GFP protein was present in the cultures, spectrophotometry should show the GFP present. qRT-PCR was used to measure the amount of mRNA produced, it measured how much of the coding gene was present in each colony. Fluorescence of qRT-PCR is proportional to the amount of mRNA.

Is there anything else we could have possibly measured to detect gene expression at the molecular level?

We could measure the actual protein level of GFP that was introduced.

Produce two graphs with error bars to represent the data collected by the two experiments.

Describe these results.

ARA-D showed signs of the most fluorescence’s and the other sugars produced 2,000,000 fluorescence’s

Do you reject or fail to reject your hypothesis?

Explain why the results contradict or agree with your original assumptions regarding the Arabinose promoter (from question 1)?  

Did DNA contamination obscure the mRNA detection in the qPCR results?  Why or why not?

Propose a new hypothesis based on the results and describe the next experiment you think should be performed?

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Answer 1

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