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

You believe that the cytosolic protein p007 is involved in

You believe that the cytosolic protein p007 is involved in mediating growth hormone-stimulated proliferation in cells. You wish to establish a cell model using your cultured fibroblasts. Provide clear explanations to the ...

Explain why nbspin psoriasis is an nbspthe cells in the

Explain why  in Psoriasis is an  the cells in the dermis and epidermis thickened. Decreased cell maturation occurs in psoriasis  as a result of? Reference/s  must be provided to make  sure the  information  you are tutor ...

Steroid hormones such as testosterone are derived from

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

Bacterial cells have only one dna molecule which is double

Bacterial cells have only one DNA molecule, which is double stranded and is replicated in essentially the same fashion as in eukaryotes. Imagine that, following the techniques of other scientists studying DNA, you create ...

Frtilized grasslands diversity 6930 and richness

Fertilized grasslands: diversity = .6930 and richness = 4 Unfertilized grassland: diversity = .7075 and richness = 10 Answer the question by the following: 1. Which grassland has the higher species richness, and is it a ...

The m and n factors are glycoproteins that are found on the

The M and N factors are glycoproteins that are found on the surface of red blood cells antigens, The M and N factors do not cause antibody reactions in human blood transfusions. People with type M blood are homozygous fo ...

The protein produced by huntingtons disease huntingtin

The protein produced by Huntington's disease (huntingtin) interferes with general transcription factor (called CBP) and prevents it from binding the promoter. CBP activates genes involved in cell survival. i) Which step ...

Fluid electrolyte and acidbase balancedescribe the

Fluid, Electrolyte and Acid/Base Balance Describe the physiological roles of sodium, potassium, calcium, chloride, and phosphates. State the term for an excess or deficiency of each electrolyte and describe the consequen ...

Assignmentessay analysis of rhetorical strategiestattoos on

Assignment Essay: Analysis of Rhetorical Strategies Tattoos on the Heart is Gregory Boyle's memoir of running Homeboy Industries and working with gang members to help them change their lives. Boyle uses various rhetorica ...

Topic genomicsquestion describe how the use of genetic

Topic: Genomics Question: Describe how the use of genetic knowledge can change the course of disease. What are the implications of the prolific use of genetic knowledge for the intervention of disease? Should there be 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

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

Describe what you learned about the impact of economic

Describe what you learned about the impact of economic, social, and demographic trends affecting the US labor environmen