Molecular Cloning: Fluorescent Protein Cloning Worksheet 2 (Exam 2)

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Questions and Answers

What is the primary purpose of using restriction enzymes such as NotI and EcoRI in genetic engineering?

To stabilize proteins

To amplify DNA sequences

To modify RNA sequences

To cut DNA at specific sequences (correct)

Which enzyme combination would result in a different pattern of DNA fragments compared to using NotI alone?

EcoRI

NotI + EcoRI (correct)

BamHI

NotI + BamHI

When expressing a eukaryotic gene in a prokaryotic cell, which of the following is a primary concern?

The presence of introns

The different temperature requirements

Lack of post-translational modifications (correct)

The size of the plasmid

If you wanted to express the husK gene in the pancreas of a eukaryotic organism, what method might you use?

Microinjecting DNA into the zygote Signup and view all the answers

Which of the following represents a possible outcome of using BamHI in a genetic experiment?

Creation of unique DNA fragments Signup and view all the answers

Which statement best describes the expected results when using both NotI and BamHI together?

A specific number of smaller fragments Signup and view all the answers

Which factor should be considered when designing a flow chart for molecular cloning?

All steps including selection and screening Signup and view all the answers

What could be a reason for using EcoRI in a molecular cloning experiment?

To create compatible sticky ends for ligation Signup and view all the answers

What is the primary function of reverse transcriptase in the context of intron removal from the husK gene?

To create DNA copies from RNA strands Signup and view all the answers

Why do prokaryotes like E.coli have difficulty with eukaryotic genes that contain introns?

They cannot splice mRNA as eukaryotes do Signup and view all the answers

How can you include non-complementary sequences at the 5' end of a PCR primer for inserting the husK gene into pCM999?

By including any arbitrary sequence as long as the 3' end is complementary Signup and view all the answers

What is the purpose of blue-white screening when using plasmids like pCM999 that express beta-galactosidase?

To differentiate E.coli containing the plasmid without the insert from those with the insert Signup and view all the answers

Which orientation is preferable for the incorporation of husK into pCM999, and why?

Forward orientation, as it ensures correct transcription Signup and view all the answers

How can a restriction digest assist in verifying the orientation of husK in pCM999?

By producing different sized fragments based on orientation Signup and view all the answers

What would happen if the husK gene is inserted incorrectly into pCM999?

It would lead to non-functional protein production Signup and view all the answers

What characteristic of the husK gene makes it essential to process it correctly before transformation into E.coli?

It contains introns that need to be removed Signup and view all the answers

What is the primary purpose of the cloning exercise described?

To clone a fluorescent protein for further study Signup and view all the answers

Which restriction enzyme is present in the vector pCM999?

EcoRI Signup and view all the answers

How far is the BamHI restriction site from the left edge of the husK gene?

150 bp Signup and view all the answers

What is the expected outcome when E.coli is successfully transformed with the husK gene?

E.coli will produce a fluorescent protein upon UV exposure Signup and view all the answers

What challenge is presented regarding the husK gene's restriction sites?

There are no convenient restriction sites adjacent to the gene Signup and view all the answers

Which method is likely required to insert the husK gene into pCM999?

Ligation after restriction digestion Signup and view all the answers

Which gene in the pCM999 vector allows for selection of successful transformants?

Kanamycin resistance gene Signup and view all the answers

What is the role of the EcoRI restriction site within the cloning process?

To create sticky ends for ligation Signup and view all the answers

Study Notes

Advanced Cloning Exercise

The goal is to clone a fluorescent protein (husk gene) from a Wild Golden Dog cell and express it in E. coli.
The husky gene is located on a chromosome section (2000 bp).
It does not have convenient restriction sites around it.
The cloning vector (pCM999) has restriction sites for EcoRI and NotI, 300 bp apart.
The goal is to insert the husk gene into pCM999.
The distance from the left edge of the husk gene to the BamHI restriction site is 150 bp.
The pCM999 plasmid normally expresses beta-galactosidase, which is used for blue-white screening to identify bacteria with the vector.
To insert the gene, PCR primers can be designed to amplify the husk gene, with the 5’ end of the PCR primer not needing to be complementary to anything, but the 3’ end must be complementary.

Cloning Strategy

Isolating the husk gene: Reverse transcriptase can be used to create DNA copies of the RNA to remove introns from eukaryotic cells. PCR can amplify the gene using primers designed to surround the gene and use specific restriction sites.
Digesting and inserting husk gene: The amplified DNA fragment and vector (pCM999) are digested with appropriate restriction enzymes, to create compatible sticky ends, and then ligated together. Then the result is transformed into E. coli with the proper antibiotic.
Screening for transformants: The transformants are selected using antibiotic resistance and blue-white screening, where bacteria that successfully took up the vector and have the husk gene express a fluorescent protein, which can be seen when exposed to UV light.
Orientation considerations: The husk gene can be inserted into the pCM999 vector in either forward or reverse orientation. A restriction digest can determine the orientation of the gene.

Additional Questions

Eukaryotic gene expression in prokaryotes concerns: Eukaryotic genes contain introns, which prokaryotic cells cannot process. Thus, using reverse-transcriptase can create DNA copies from the RNA to remove introns from eukaryotic mRNA.
Eukaryotic expression: To express a eukaryotic gene in a eukaryotic cell, direct injection into the zygote is one option. Factors like specific location (pancreas) and appropriate promoters influence targeted expression.
Enzyme(s) to detect orientation: Specific restriction enzymes can be used to determine the orientation of the cloned gene within the vector.

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Description

This quiz covers the advanced cloning exercise involving the husk gene from Wild Golden Dog cells and its expression in E. coli. You'll learn about the cloning strategy, including PCR primer design and the blue-white screening process using the pCM999 vector. Test your knowledge on the techniques and principles of molecular cloning.