botc lab 10

plate observation (-DNA, LB)

An even lawn of bacteria is present on this plate. The lawn appears off-white.

From your results, can you tell if these bacteria are ampicillin resistant by looking at them on the LB plate? Explain your answer.

No. You cannot tell if the bacteria are ampicillin resistant just by looking at them. Both types of bacteria (those that are ampicillin resistant and those that are ampicillin sensitive) look similar when cultured.

in this lab (pglo transformation) you will perform a procedure known as a

genetic transformation

process of moving genes from one organism to another with the aid of a ___

plasmid

list all observable traits or characteristics that can be described (6)

1. number of colonies; 2. size of... largest colony, smallest colony, majority colonies; 3. color of the colonies; 4. distribution of the colonies on the plate; 5. visible appearance when viewed with uv lights; 6. the ability of the cells to live and reproduce in the presence of an antibiotic such as ampicillin

What is meant by control plate? What purpose does a control serve?

A control plate is a guide that is used to help you interpret the experimental results. In this experiment, both -DNA plates are control plates. The LB/amp control plate can be compared to the LB/amp +DNA plate. This comparison shows that genetic transformation produces bacterial colonies that can grow on ampicillin (due to the uptake of the pGLO plasmid and the expression of the ampicillin resistance gene). The -DNA/LB control plate can be compared to any of the LB/amp plates to show that plasmid uptake is required for the growth in the presence of ampicillin. The -DNA LB/amp plate shows that the starter culture does not grow on the LB/amp plate. Without this control one would not know if the colonies on the LB/amp +DNA plate were really transform ants.

Describe the evidence that indicates whether your attempt at performing a genetic transformation was successful or not successful.

A successful experiment will be represented by the presence of colonies on the +DNA LB/amp and +DNA LB/amp/ara plates and the absence of colonies on the -DNA LB/amp plate. Moreover, the colonies on the LB/amp/ara plate should fluoresce green.; An unsuccessful experiment will show an absence of colonies on the +DNA LB/amp and +DNA LB/amp/ara plates. This could be a result of not adding a loopful of plasmid to the +DNA tube or not adding a colony of bacteria to the +DNA tube.

On which of the plates would you expect to find bacteria most like the original untransformed E. coli colonies you initially observed? Explain your prediction.

Bacteria which resemble the non-transformed E. coli will be found on the LB/-DNA plate. These bacteria were removed from the starter plate, did not have any plasmid added to them, and were replated on an LB plate. Thus, they are virtually identical to the non-transformed starter E. coli.

Very often an organism's traits are caused by a combination of its genes and the environment it lives in. Think about the green color you saw in the genetically transformed bacteria: What advantage would there be for an organism to be able to turn on or off particular genes in response to certain conditions?

Gene regulation allows for adaptation to differing conditions and prevents wasteful overproduction of unneeded proteins. Good examples of highly regulated genes are the enzymes which break down carbohydrate food sources. If the sugar arabinose is present in the growth medium it is beneficial for bacteria to produce the enzymes necessary to catabolize the sugar source. Conversely, if arabinose is not present in the nutrient media, it would be very energetically wasteful to produce the enzymes to break down arabinose.

plate observation (+DNA, LB/amp/ara)

Many transformed colonies of bacteria (optionally ~75). Colonies appear white when exposed to room light but fluoresce bright green when exposed to UV light.

plate observation (+DNA, LB/amp)

Many transformed colonies of bacteria (optionally ~75). Colonies appear white.

plate observation (-DNA, LB/amp)

No bacterial growth present on this plate.

Which plates should be compared to determine if any genetic transformation has occurred? Why?

The LB/amp -DNA and the LB/amp +DNA plates should be directly compared. Cells which were not treated with DNA (-DNA) should not be expressing the ampicillin resistance gene and will not grow on the LB/amp plates. Cells which were treated with DNA (+DNA) should contain the pGLO plasmid and should express the ampicillin resistance gene—the corresponding LB/amp plate will contain transformed bacterial colonies.

What color are the bacteria?

The bacteria on the +DNA LB/amp plate and the -DNA LB plates should be whitish. The bacteria on the +DNA pGLO LB/amp/ara plate should appear whitish when exposed to normal, room lighting, but fluoresce bright green upon exposure to the long-wave UV light.

How would you change the bacteria's environment to best tell if they are ampicillin resistant?

The best test would be to take some of the bacteria growing on the LB plate and streak them on an LB/amp plate. If the bacteria are viable on the LB/amp plate, then they are resistant to ampicillin. If no bacterial colonies survive, then they were not ampicillin resistant (they were ampicillin sensitive).

If the genetically transformed cells have acquired the ability to live in the presence of the antibiotic ampicillin, then what can be inferred about the other genes on the plasmid that were involved in your transformation procedure?

The plasmid must express a gene for ampicillin resistance (the protein product of the bla gene codes for beta-lactamase, the protein that breaks down ampicillin).

Very often an organism's traits are caused by a combination of its genes and the environment it lives in. Think about the green color you saw in the genetically transformed bacteria: a. What two factors must be present in the bacteria's environment for you to see the green color? (Hint: one factor is in the plate and the other factor is in how you look at the bacteria).

The sugar arabinose in the agarose plate is needed to turn on the expression of the GFP gene. The UV light is necessary to cause the GFP protein within the bacteria to fluoresce.

Very often an organism's traits are caused by a combination of its genes and the environment it lives in. Think about the green color you saw in the genetically transformed bacteria: b. What do you think each of the two environmental factors you listed above are doing to cause the genetically transformed bacteria turn green?

The sugar arabinose turns on expression of the GFP gene by binding to a regulatory protein, araC。 The araC protein acts as a repressor and binds to the promoter of the GFP gene, preventing its transcription. When arabinose is present, it binds to araC protein, consequently changing the conformation of araC which facilitates transcription of the gene by RNA polymerase (araC protein cannot bind the promoter of GFP when it is bound with arabinose - therefore the RNA polymerase is able to transcribe the GFP gene). Exposure to UV light causes GFP to resonate, thereby giving off energy in the form of green light.

If there are any genetically transformed bacterial cells, on which plate(s) would they most likely be located? Explain your prediction.

The transformed cells are found on the LB/amp and LB/amp/ara plates. Genetically transformed cells have taken up the pGLO plasmid which expresses the ampicillin resistance gene—these cells can survive on the plates which contain ampicillin.

How much bacterial growth do you see on each, relatively speaking?

There should be multiple colonies on both the LB/amp and LB/amp/ara plates that received the pGLO plasmid (optionally ~ 75 colonies). There should be no growth on the LB/amp -DNA plate. There should be a lawn of bacteria on the LB/ -DNA plate.

Count how many bacterial colonies there are on each plate (the spots you see).

There should be optionally ~ 75 bacterial colonies on the two +DNA plates. The lawn of bacteria on the LB plate contains an even spread of bacteria and individual colonies can't be counted.

the gene for the gfp can be switched on in transformed cells by adding the sugar, ____, to the cells' nutrient nutrient medium

arabinose

genetic transformation

change caused by genes and it involves the insertion of a gene or genes into an organism in order to change the organism's trait(s)

pglo plasmid encodes the gene for the ___ and a gene for resistance to the antibiotic ampicillin

gfp

gfp

green fluorescent protein

___ ___ usually contains genes for one or more traits that may be beneficial to bacterial survival

plasmid dna

the recent occurrence of bacterial resistance to antibiotics is due to the transmission of ___

plasmids