1. You are planning to study a gene ydeH from E.coli that encode for putative diguanylate...
1. You are planning to study a gene ydeH from E.coli that encode for putative diguanylate cyclase activity (synthesis of a second messanger c-di-GMP). The in vitro enzymatic assay requires incubation of protein with GTP. List the steps involve in purification of protein. (hint : you need to clone the gene, express and purify the protein for in vitro assays.)
1a. Find the sequence of gene: Using NCBI or other database
2b. Design a strategy to clone and express the gene
- How will you clone the gene using PCR.?
- Design primers (sequence)
- Clone it in a plasmid (you can choose a plasmid from pET21b, pET28a, pUC19, pBAD24
- how will you select and screen the colonies?
- how will you express the protein?
- how will the protein be purified?
Homework Answers
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1. You are planning to study a gene ydeH from E.coli
that encode for putative diguanylate...
This sequence encodes a protein that you wish to study further by cloning the gene. The...
This sequence encodes a protein that you wish to study further by cloning the gene. The protein of interest has a molecular weight of at least 10 kDa. The sequence was sequenced by a primer walking approach, but unfortunately the correct order of the sequence traces was lost (really bad lab notebook skills). Trace 1 aacgagttaaggagccagcgtaccttcgcaccgccatacatgaattttcttggctttttctatgtggatggcaatagtctagagtcggacctgcaggcatgcaag Trace 2 cagcctgttagtaggtcttactgagtcgggcgccgaattcgagctcggtacccggggatccatgagtgggcgccagttattcgtactattgggaggtcc Trace 3 ccgggtattttgtattcaatattgaataaggaatttttcatgcagagaaaaggatgtttacggctcgagcgcactcgcacatataactgtcggcagaaacgagttaaggagc Trace 4 tactattgggaggtccaaatggttttacgggagttgcactgggcgaatgctggagctattacgattcgtttaacatttccatatcgaattctcagacgactccgaatccgggtatttt Trace 5 cctgcaggcatgcaagcttggcataggtcagttcatccagggtgatgggtgtatcgtttcaatgacccgattcggaacg Answer the following-- Determine the correct order of the sequence traces and...
You wish to clone the human cnr1 gene into the DH5 alpha strain of E.coli. Design...
You wish to clone the human cnr1 gene into the DH5 alpha strain of E.coli. Design an outline for this project based on the PCR, Restriction digestion and complementation labs. Make sure you explain how you will acquire the gene sequence and the type of plasmid you plan on using.
You are assigned to purify a protein from an E.coli cell lysate. The following information is...
You are assigned to purify a protein from an E.coli cell lysate. The following information is provided to you about the target protein. The protein has an N-terminal 6X histidine tag (His-tag) attached to it The protein is very stable in PBS (Phosphate Buffered Saline) buffer that you need to use as the storage buffer after purification It is an enzyme that will be used as a potential drug. You have the amino acid sequence of the protein. Based on...
1) Imagine that you are interested in studying the enzyme dihydrofolate reductase (DHFR) from Mycobacterium tuberculosis...
1) Imagine that you are interested in studying the enzyme dihydrofolate reductase (DHFR) from Mycobacterium tuberculosis (this enzyme is a potential drug target). The amino acid sequence of the protein is: MTMVGCIWAQATSGVIGRGGDIPWRLPEDQAHFREITMGHTIVMGRRTWDSLPAKVRPLPGRRNWL SRQADFMASGAEWGSLEEALTSPETWVIGGGQVYALALPYATRCEVTEVDIGLPREAGDALAPVLD ETWRGETGEWRFSRSGLRYRLYSYHRS The DNA sequence within the M. tuberculosis genome that codes for the protein is: ATGACGATGGTGGGGCTGATCTGGGCTCAAGCGACATCGGGTGTCATCGGCCGCGGCGGCGACATCCCCT GGCGCTTGCCCGAGGACCAGGCGCATTTCCGGGAGATCACCATGGGGCACACGATCGTGATGGGCCGGCG CACATGGGATTCGCTGCCGGCTAAAGTCCGGCCGCTGCCCGGCCGGCGAAATGTCGTACTGAGCCGCCAA GCTGACTTTATGGCCAGCGGGGCTGAGGTTGTCGGTTCACTCGAGGAGGCGCTGACCAGCCCGGAGACGT GGGTGATCGGAGGCGGACAAGTCTATGCGCTGGCGCTGCCGTACGCGACCAGATGTGAGGTTACCGAGGT CGACATCGGCCTGCCGCGCGAAGCCGGTGACGCGCTGGCCCCCGTGCTGGACGAGACATGGCGGGGCGAG ACGGGGGAGTGGCGCTTCAGCCGGTCCGGGTTGCGGTACCGGTTGTACAGCTACCACCGCTCATGA Assume that you have been given a sample of the bacterial genomic DNA, restriction enzymes Ndel and BamHi, and a plasmid from Novagen called PET-28b....
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Draw (or arrange) only the items you need from the Lab Materials Page (picture below) in...
Draw (or arrange) only the items you need from
the Lab Materials Page (picture below) in the
order you would use them to make AND screen a gene library.
You will ‘draw’ the steps that use the Lab Materials in the correct
order to create and screen a gene library which has the assigned
gene clone. You will need to add some connecting words and phrases
to make all the steps for making your library clear.
The genomic gene clone...
Molecular Bio lab. HELP!! Here is the first part: the sequence traces and the entire sequence....
Molecular Bio lab. HELP!!
Here is the first part: the sequence traces and the entire
sequence. i just need the last 3 tasks. i color coded the ends so
you can see where it overlaps and connects
In the files section for your group there is a simulated output from an automated DNA sequencer using a variation of the classic Sanger method. (If you want to print it, it is formatted for legal sized paper.) This sequence encodes a protein...
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1. You are studying phage transduction and trying to deliver a kanamycin resistance gene from one...
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1) Imagine that you are interested in studying the enzyme dihydrofolate reductase (DHFR) from Mycobacterium tuberculosis (this enzyme is a potential drug target). The amino acid sequence of the protein is: MTMVGCIWAQATSGVIGRGGDIPWRLPEDQAHFREITMGHTIVMGRRTWDSLPAKVRPLPGRRNWL SRQADFMASGAEWGSLEEALTSPETWVIGGGQVYALALPYATRCEVTEVDIGLPREAGDALAPVLD ETWRGETGEWRFSRSGLRYRLYSYHRS The DNA sequence within the M. tuberculosis genome that codes for the protein is: ATGACGATGGTGGGGCTGATCTGGGCTCAAGCGACATCGGGTGTCATCGGCCGCGGCGGCGACATCCCCT GGCGCTTGCCCGAGGACCAGGCGCATTTCCGGGAGATCACCATGGGGCACACGATCGTGATGGGCCGGCG CACATGGGATTCGCTGCCGGCTAAAGTCCGGCCGCTGCCCGGCCGGCGAAATGTCGTACTGAGCCGCCAA GCTGACTTTATGGCCAGCGGGGCTGAGGTTGTCGGTTCACTCGAGGAGGCGCTGACCAGCCCGGAGACGT GGGTGATCGGAGGCGGACAAGTCTATGCGCTGGCGCTGCCGTACGCGACCAGATGTGAGGTTACCGAGGT CGACATCGGCCTGCCGCGCGAAGCCGGTGACGCGCTGGCCCCCGTGCTGGACGAGACATGGCGGGGCGAG ACGGGGGAGTGGCGCTTCAGCCGGTCCGGGTTGCGGTACCGGTTGTACAGCTACCACCGCTCATGA Assume that you have been given a sample of the bacterial genomic DNA, restriction enzymes Ndel and BamHi, and a plasmid from Novagen called PET-28b....
Draw (or arrange) only the items you need from
the Lab Materials Page (picture below) in the
order you would use them to make AND screen a gene library.
You will ‘draw’ the steps that use the Lab Materials in the correct
order to create and screen a gene library which has the assigned
gene clone. You will need to add some connecting words and phrases
to make all the steps for making your library clear.
The genomic gene clone...
Molecular Bio lab. HELP!!
Here is the first part: the sequence traces and the entire
sequence. i just need the last 3 tasks. i color coded the ends so
you can see where it overlaps and connects
In the files section for your group there is a simulated output from an automated DNA sequencer using a variation of the classic Sanger method. (If you want to print it, it is formatted for legal sized paper.) This sequence encodes a protein...
1. Describe the functions of the following reagents in extraction of DNA from corn meal: proteinase K; guanidine HCI; SDS 2. Why is the ratio of the OD at 260 and 280 nm used to estimate DNA purity? 3. In one paragraph, summarize basic principles of PCR technique in your own words. List all the reagents you will need to perform a PCR experiment. Does this method tell you what genetic modifications were made? If yes, describe how you can...
1. You are studying phage transduction and trying to deliver a kanamycin resistance gene from one strain of bacteria to another using phage. The kanR gene is FAR away from the lambda prophage in your bacterium. Choose which type of transduction should you use for your experiment and explain why. a. lambda phage, specialized transduction b. lambda phage, conjugation C. T4 phage, generalized transduction d. A different phage with mitomycin C induction 2. A pig is infected with two subtypes...
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