Term
- What is suicide plasmid? How can you disrupt a gene, or construct a lacZ fusion into a locus using suicide plasmid? (Hint: use a piece of target DNA sequence)
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Definition
A vector incapable of replicating in a specific host. Maintenance of the selected marker necessitates integration into the chromosome.A suicide plasmid is one which cannot normally replicate in the host. It is therefore lost from the cells during growth. Suicide plasmids have been used to introduce e.g. transposons into cells, or an interrupted version of a gene (usually with an antibiotic resistance cassette) that will replace the wild type allelle by homologous recombination. Suicide plasmids can be propagated in certain strains that supply a factor required for the replication of the suicide plasmid in trans. Other techniques have to some extent replaced suicide plasmids. A vector incapable of replicating in a specific host. Maintenance of the selected marker necessitates integration into the chromosome.
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Term
What is the molecular structure of crossover?
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Definition
During recombination molecular crossover is defined as the breakage and rejoining of 2DNA molecules knew new combination. The position of the bases are shown only where the mutation has occured (-)= mutation, (+) =wild-type, note that while one crossover between a region of 2 mutations will make a recombinant Type. 2 crossovers will create a parental type. Odd # of crossover = recombinant type and even #= parental type.
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Term
How can you disrupt a gene, or construct a lacZ fusion into a locus using a linear DNA carrying an ampicillin-resistant cassette?
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Definition
In DNA you should have origin. This origin comes from corE1 plasmid. This plasmid thus is not naturally occurring and is manmade. So the pUC19 origin of replication (oriV) is derived from the corE1 plasmid.
It contains a DNA fragment…a gene….called Bla gene that encodes lactamase (Beta-lactamase that can open/cut a bond in ampicillin, thus destroying the ampicillin molecule. Ampicillin inhibits synthesis of cell wall. If a bacteria can synthesize beta-lactamase that can refute the activity of ampicillin, then the bacteria can survive in a media containing ampicillin).
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Term
Give an example to explain which step that can be regulated in a protein production? In bacteria, which step is most commonly regulated? Why?
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Definition
In bnacteria the step that is most easily regulated is transcription, this is expemplified in the lac operon. In which lacI causes repression of the lac operon until the proper inducer, lactose, not in the presents of glucose is present. The lactose will bind to the inhibitor, lacI, and Cap and Camp protines will then be aLLOWS to bind to the promoter and transcription begins.
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Term
Cell growth was measured in a medium that has glucose and lactose in it and the results are shown in the graph below. Provide an explanation for what is happening to bacterial gene regulation with regard to cellular response to sugar source (glucose, lactose, galactose, and arabinose) at the three stages (A, B, and C).
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Definition
In the presents of glucose the lacI repressor is still bound to the operator, because it is more adventagious for the cell to use glucose over lactose. When the graph hits point B the glucose has been used up and the cell starts to activate the lac operon, thus lactose causes the lacI to leave the operator and the CAP and Camp complex is alloed to bind and transcription begins. And thus in point C the growth of the bacteria begins again using lactos instead of glucose.[image]
“A” is when the cells are utilizing a monosaccharide (glucose) utillzing cAMP so cAMP is low causing the activator (CAP protein) to not bind so even though there is a presence of lactose and galactose in the medium they will not be transcribed. After the monosaccharide has been depleted, cells will not grow and cAMP will increase which is seen in “B”, a point “C” is when cAMP binds with CAP to make a complex for an activator and since the there is a presensce of galactose and lactose, galR and lacI is removed (induced) so they are transcribed.
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Term
You hypothesize that a system may regulate expression of several genes. You want to find out what these genes are. Design a cDNA microarray experiment, to identify these genes. What procedure would you perform? Describe the A,B,C steps in this procedure.
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Definition
Engineered plasmids in bacterial genetic analysis=recombinant plasmid. Amplify all the gene from a chromosomal DNA which containing unknown function of these genes and introduce them to the plasmid which have the promoter and express the gene. when the gene express you can observe and identify the functions of these genes.
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Term
What do you think bacteria may take advantage of organization of operon in gene regulation? Please give examples to support your opinion.
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Definition
Bacteria may take advantage of organization of an operon in gene regulation in order to use the simplest carbon source. Using the organization of an operon organisms can either express certain genes under different environmental conditions.
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Term
Design an experiment to show an operator in an operon is a cis-acting factor, and describe the steps in this procedure.
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Definition
An experiment to show that the operator in an operon is a cis-acting factor is to induce mutation the operator and see if the genes are still transcribed. If they are not transcribed if the operon is mutated, then it is cis-acting.
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Term
wo mutations took place in (1) a gene, (2) two neighboring genes. Describe how you distinguish these two possibilities using a complementation analysis.
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Definition
Using complementation analysis, we should determine if the mutation is recessive to the wild type. If the mutation is recessive for wild type, it can be used for complentation analysis. If the mutation complements each other, it can affect different genes (neighboring genes). If the recessive mutations cannot complement each other they affect the function of the same gene.
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Term
- What is a super repressor LacIS? Why are super-repressor mutants dominant to a wild type repressor?
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Definition
A super repressor (lacIS) is a repressor that binds to an operator and is non-inducible so it cannot be removed. These are dominant because they will bind and never be removed, even if the wild type is attached to one, once it is removed the lacIS may attach and never be removed.
The super repressor mutation result in a changed lac I. They are different from the lac I wild type protein. This gene prevents inducer (lactose or IPDG) from ever binding to the operator. lacs is a dominant phenotype, which in the present of wild type, will still exhibit the mutation. However recessive phenotype can be complimentary to the wild type.
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Term
What is lacOc? What is the phenotype of this mutant? And why?
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Definition
acOc is an operator that is mutated causing lacI repressor the inability to bind causing it to be constitutive; phenotype is blue on an x-gal plate regardless of the presence of lactose since it’s always being transcribed.
Lac O is DNA sequence for operator that bind to the repressor. So when it is mutated, it will have the same phenotype as lac I, resulting in constitutive expression. This means the lac operon is transcribe regardless of the presence of lactose.
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Term
t will be important to explain a phenotype that is similar to one of those in Table12.1 (Textbook).
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Definition
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Term
Please explain the regulator that controls global gene regulation of utilization of different sugars in E. coli.
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Definition
The regulators that control the gene regulation for utilization of different sugars in E coli are the CAP and Camp catabolite repression proteins. The presents of glucose, the most use ful =sugar causes there to be low amount of cAMP. When glucose is absent cAMP levels rise, they will bind to CAP protein which activates the lac transcription. Thus cAMP and glucose are inversely related if one is high the other is low.
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Term
What is a regulon? Give an example. Why are regulons important?
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Definition
Regulon is a collection of genes or operons under regulation by the same regulatory protein. An example is the lac operon being controlled be the CAP protein. because you dont want to express the lac operon all the time. unless there is lactose in the medium.
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Term
Please give examples to explain why and how a bacterium triggers global gene regulation and when would it be useful for a cell?
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Definition
Bacteria must be able to adapt to the environment around them, nutrition changes very rapidly and the bacteria must be able to fend off starvation, temperature varies widely, water vaerie. Not only do conditions change they can change abruptly so the bacteria must be able to deal and cope with this abrupt change so that they do not die. They need to be able to transcribe and translate many proteins very fast, and what is faster than having many proteins that are essential for temperature change, or carbon source on one operon. Thus these operons are all regulated by one regulon, and when the right conditions occur the operon is activated and the proteins are made that can save the life of the cell.
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