Ask Question, Ask an Expert

+1-415-315-9853

info@mywordsolution.com

Ask Biology Expert

Engineering a recombinant plasmid DNA(like GST- preferably use other than GST)to over-express the protein encoded by the MsrB gene from Drosophila, by cloning it into the pGEX vector. the purpose of this assignment is to allow to design our own similar approach for overexpression of a recombinant protein of our own choosing.

1-select one Drosophila gene that you would like to use for this project. FlyBase.org is a database of Drosophila genes and is therefore a good place to start, however we can obtain our gene from another source if we prefer.
2-find the DNA sequence for the open-reading frame (ORF) of our selected gene. this is the portion of the mRNA that codes for the amino acids. It will start with an ATG(AUG) 'start codin, and end with one of the three 'stop' codons (TAG,TAA,TGA), often, the complete gene sequence will be shown with the exon highlighted.
3-design PCR primers between 25-30 nucleotides in length that can be used to amplify the cDNA and allow cloning into the pGEX-4T-2 vector. as shown on the map of the pGEX vector, we can use BamHI,EcoRI,SmaI,SalI,XhoI or NotI for cloning.
4-show the cDNA sequence and corresponding primer sequence for both the upstream and downstream primers. be very clear about any base changes in the primer that are needed to create the restriction site and or maintain the correct reading frame.
5-determine the amino acid sequence that precedes the Met start codon after the fusion protein is cleaved with thrombin.
IMPORTANT POINTS TO KEEP IN MIND:
1-the cDNA must be inserted in the correct orientation. the 5' end must be closest to the thrombin cleavage site.
2-our PCR amplicon cannot contain a recognition site for either of the enzymes that we have chosen for cloning (why not?)
3-we must maintain the proper reading frame. note how bases can be added or delete from the 5' upstream primer as needed.
4-try to clone the cDNA as close to the start codon as possible.
5-we do not have to worry about the reading frame for the downstream primer, but be sure that the stop codon of the cDNA is retained.

Biology, Academics

  • Category:- Biology
  • Reference No.:- M928419

Have any Question? 


Related Questions in Biology

Biology questions1 what were the characteristics of the pea

Biology questions 1. What were the characteristics of the Pea plant that enabled Mendel to do his experiments? 2. How does probability and chance play an important role in genetics? 3. Explain the difference between gene ...

Two questions to be answered1 government and the arts

TWO Questions to be answered 1. "Government and the Arts; Abstract Expressionism and Music" Please respond to one of the following, using sources under the Explore heading as the basis of your response: • Examine the U.S ...

What fundamental assumption underlies the concept of an

What fundamental assumption underlies the concept of an ocean commons?

Steroid hormones such as testosterone are derived from

Steroid hormones, such as testosterone, are derived from cholesterol. What type of macromolecule are they?

How might 32p and 35s be used to demonstrate that the

How might 32P and 35S be used to demonstrate that the transforming principle is DNA? Briefly outline an experiment that would show that DNA rather than protein is the transforming principle.

Given what you know about how natural selection works

Given what you know about how Natural Selection works, explain why Sickle cell disease hasn't been eradicated from the population.

Answer the following questionwhat is the domain and kingdom

Answer the following question: What is the domain and kingdom for an organism that has a nucleus, is not motile, and does not perform photosynthesis? What is taxonomy? What role does this serve? How does taxonomy assist ...

Describe the performance of a urinalysis including

Describe the performance of a urinalysis, including equipment.

Imagine that you had 11000 of the worlds gross domestic

Imagine that you had 1/1000 of the worlds gross domestic product available at your disposal for conservation biology product? Is it a sound conservation decision to protect just 4% of the earth's living habitat? Why or w ...

Where have you observed an ecosystem change over time how

Where have you observed an ecosystem change over time? How would you describe the biodiversity - in terms of species richness and evenness - of the place and how the change affected biodiversity? What are the positive an ...

  • 4,153,160 Questions Asked
  • 13,132 Experts
  • 2,558,936 Questions Answered

Ask Experts for help!!

Looking for Assignment Help?

Start excelling in your Courses, Get help with Assignment

Write us your full requirement for evaluation and you will receive response within 20 minutes turnaround time.

Ask Now Help with Problems, Get a Best Answer

WalMart Identification of theory and critical discussion

Drawing on the prescribed text and/or relevant academic literature, produce a paper which discusses the nature of group

Section onea in an atwood machine suppose two objects of

SECTION ONE (a) In an Atwood Machine, suppose two objects of unequal mass are hung vertically over a frictionless

Part 1you work in hr for a company that operates a factory

Part 1: You work in HR for a company that operates a factory manufacturing fiberglass. There are several hundred empl

Details on advanced accounting paperthis paper is intended

DETAILS ON ADVANCED ACCOUNTING PAPER This paper is intended for students to apply the theoretical knowledge around ac

Create a provider database and related reports and queries

Create a provider database and related reports and queries to capture contact information for potential PC component pro