Explain how to make a successful plasmid construct that can knock out your intended target gene/homolog?

Expert Solution

Answer: Plasmid is autonomously replicating extrachromosomal DNA. Natural plasmids are isolated from the bacterial cell and modified artificially to get the desired plasmid. The plasmid is used as a vector to transfer a gene of interest into the target cell. Plasmids vector are used to knock out specific genes in organisms.

A successful plasmid construct can be made that can knock out your intended target gene/homolog by integrating the gene sequence (knocked out) that has homology at the flank region. Homology in the flank region enables homologous recombination. Homologous recombination causes the wild-type gene to be replaced by the knocked-out gene integrated into the plasmid (also called recombineering). The plasmid has to be designed artificially. The plasmid should have an origin of replication for the formation of copies; an antibiotic resistance gene for selection; and a recognition site for gene integration. Each arm of the target gene should have a minimum of two kilobases (kb) pairs of nucleotides for the homologous recombination. However, for successful recombination six to 10 kb is desirable. PCR methods are used for generating copies of the plasmid vector. The gene sequence to be replaced with the original wild-type gene should have a marker gene for screening out successful recombinant cells.

gladiator answered 2 years ago

Make a diagram to explain how a human gene can be cloned into a bacterial plasmid....

Make a diagram to explain how a human gene can be cloned into a bacterial plasmid. You should represent the events that need to occur in order to: Clone the insulin gene into a bacterial plasmid, Transform the plasmid into E. coli Include definitions or descriptions of the following terms/components: Restriction enzyme(s) Ligase Plasmid DNA Human mRNA (the mRNA for insulin) Reverse transcriptase Transformation E. coli marker genes (an antibiotic resistance gene) Cloning vector

If an embryo had knock-out mutations in both a gap gene and a segment polarity gene,...

If an embryo had knock-out mutations in both a gap gene and a segment polarity gene, determine which mutant phenotype you would expect to see. Explain your reasoning?

Design an experiment to knock out a certain gene of interest in the human genome. Outline...

Design an experiment to knock out a certain gene of interest in the human genome. Outline the experiment, explaining the methods you would use, and also indicate how you would verify your experiment was successful at each step.

You are inserting your gene of interest into the Lac Z gene in a plasmid also...

You are inserting your gene of interest into the Lac Z gene in a plasmid also containing a tetracycline resistant gene. You plate your transformed bacteria on media containing tetracyline and X-gal. Which of the following results would indicate a clone with recombinant plasmids? Select one: a. A blue colony on the tetracyline plates b. A clone which did not grow on the tetracycline plates c. A red colony on the tetracyline plates d. A white colony on the tetracyline...

3) How can you use PCR to clone a 2000bp gene into a plasmid vector?

You want to use Cre/loxP system for targeted gene expression instead of knock-out. Briefly describe how...

You want to use Cre/loxP system for targeted gene expression instead of knock-out. Briefly describe how this can be accomplished. (3-4 sentences)

Describe in detail how to clone your “gene of interest” into a bacterial plasmid using EcoRI...

Describe in detail how to clone your “gene of interest” into a bacterial plasmid using EcoRI restriction enzyme and how to generate a large quantity of the DNA using competent E. coli. Why do you use competent bacteria for this?

Explain in your words, that the intention is to make a budget and how it can...

Explain in your words, that the intention is to make a budget and how it can be the help service for the administration of health entities

The AmpR gene on plasmid pGLO gives cell resistance to Ampicillin. Explain what that means and...

The AmpR gene on plasmid pGLO gives cell resistance to Ampicillin. Explain what that means and how this gene provides that.

How would you clone the Eukaryotic gene into the plasmid How would you transform bacteria with...

How would you clone the Eukaryotic gene into the plasmid How would you transform bacteria with that engineered plasmid How would you select for the bacteria that will take up the plasmid with integrated Eukaryotic gene

Question