Sample 6a Transformation Lab

 

 

Lab 6A – Bacterial Transformation & Ampicillin Resistance

 

 

Introduction:
Bacterial transformation occurs when a bacterial cell takes up foreign DNA and incorporates it into its own DNA. This transformation usually occurs within plasmids, which are small circular DNA molecules separate from its chromosome. There can be 10 to 200 copies of the same plasmid within a cell. These plasmids may replicate when the chromosome does, or they may replicate independently. Each plasmid contains from 1,000 to 200,000 base pairs. Certain plasmids, called R plasmids, carry the gene for resistance to antibiotics such as ampicillin, which is used in this lab.

Plasmids function in transformation in two different ways. They can transfer genes that occur naturally within them, or they can act as vectors for introducing foreign DNA. Restriction enzymes can be used to cut foreign DNA and insert it into the plasmid vectors. The bacteria used in this lab were Escherichia coli (E. coli). It was ideal for this transformation study because it can be easily grown in Luria broth or on agar, and it has a relatively small genome of about five million base pairs.

Transformation is not the only method of DNA transfer within bacteria. Conjugation is a DNA transfer that occurs between two bacterial cells. A bridge is formed between the two cells and genetic information is traded. In transduction, a virus is used to transfer foreign DNA into a bacterial cell.

Hypothesis:
The transformed E. coli with the ampicillin resistance gene will be able to grow in the ampicillin plates, but the non-transformed E. coli will not.

Materials:
The materials needed for this lab were 2 sterile test tubes, 500 μL of ice cold 0.05M CaCl2, E. coli bacteria cultures, a sterile inoculating loop, a sterile micropipette, 10 μL of pAMP solution, a timer, ice, a water bath, 500 μL of Luria broth, a spreading rod, 4 plates: 2 ampicillin+ and 2 ampicillin – , and an incubator.

 

Methods:
One sterile tube was labeled “+” and the other “-“. A sterile micropipette was used to transfer 250 μL of ice cold 0.05M CaCl2 to each tube. A large colony of E. coli was transferred with an inoculating loop to each tube. The suspension was then mixed by repeatedly drawing and emptying a sterile micropipette. 10μL of pAMP solution was added to the cell suspension in the tube marked “+” and mixed by tapping the tube. Both tubes were immediately put on ice for 15 minutes and then soaked in a 42° C water bath for 90 seconds. The tubes were then returned to ice for another 2 minutes.

After the heat shock, 250 μL of Luria broth were added to each tube. The tubes were mixed by tapping. Two plates of ampicillin + agar were labeled LB/AMP+ and LB/AMP-. The two plates of ampicillin- agar were labeled LB+ and LB-. 100 μL of the cell suspension in the “+” tube were placed on the LB+ and the LB/AMP+ plates. 100μL of the cell suspension in the “-” tube were added to the LB- and the LB/AMP- plates. These were spread with a spreading rod that was sterilized by passing it over a flame after each use. The plates were allowed to sit for several minutes and then incubated over night inverted at 37° C.

 

Results:

 

 

 

LB+

(Positive Control)

 

LB-

(Positive Control)

 

LB/AMP+ (Experimental)

 

LB/AMP- (Experimental)

 

Bacterial Growth

lawnlawn3 coloniesNo growth

 

Questions:
1. Compare and contrast the number of colonies on each of the following pairs of plates. What does each pair of results tell you about the experiment?
LB+ and LB- Both of these plates had a lawn of bacteria. This proves that the bacteria are capable of growing on the agar and that there was nothing preventing growth beside the ampicillin.

LB/AMP- and LB/AMP+ The LB/AMP- had no growth, but the LB/AMP+ had small growth. This shows that the bacteria was transformed and developed a resistance to ampicillin.

LB/AMP+ and LB+ The LB/AMP+ had less growth than the LB+. This shows that the transformation was not completely effective and only transformed some of the most competent bacterial cells.

 

2.  Total mass of pAMP used = 0.05 μg

 

Total volume of cell suspension = 510 μL

 

Fraction of cell suspension spread on the plates = 0.196

 

Mass of pAMP in cell suspension = 0.0098

 

Number of colonies per μg of plasmid = 0.0294

3. What factors might influence the transformation efficiency? Explain the effect of each you mention.
Transformation efficiency could be affected by the size of the colony added to the solution. In a larger colony the efficiency would increase because there would be more receptive cells. Another factor would b the amount of pAMP added. The more pAMP added, the higher the efficiency. The amount of Luria broth added could also affect efficiency. If the amount of Luria broth was increased, the efficiency would decrease.

 

Error Analysis:
This lab had several steps, each giving the potential for error. All of the measurements had to be precise and accurate, and the heat shock timing was also a very complicated procedure. Error in this lab could have been caused by the concentration of the CaCl2 due to the fact that most of it was frozen.

 

Discussion and Conclusion:
The bacteria treated with the pAMP solution developed a resistance to ampicillin and were able to grow on the ampicillin+ plate. Those that were not treated with the pAMP were not able to grow on this medium. The plates with no ampicillin served as a control to show how the bacteria would look in normal conditions. Transformation is never fully effective, Only cells that are competent enough are able to take up the foreign DNA. Therefore, the ampicillin + plates showed less growth than the control plate.

Pzsol Viruses

 

Viruses

Answer Key:

 

1) virus
2) virology
3) stanley
4) mosaic
5) crystallization
6) metabolism
7) antiviral
8) nonliving
9) capsid
10) glycoprotein
11) genome
12) transcriptase
13) envelope
14) spikes
15) nanometer
16) replicate
17) protein
18) nucleic
19) hiv
20) immunodeficiency
21) icosahedron
22) helix
23) host
24) adenovirus
25) retrovirus
26) viroid
27) prion
28) intracellular
29) bacteriophage
30) escherichia
31) collar
32) tail
33) lytic
34) virulent
35) assembly
36) lysis
37) attachment
38) lysogenic
39) temperate
40) prophage
41) injection
42) inactivated
43) attenuated
44) smallpox
45) measles
46) influenza
47) jungles

 

Pzsol Worms

 

Worms
Answer Key:
 

1)platyhelminthesphylum of flatworms
2)threenumber of body layers in worms
3)bilateralsymmetry f flatworms
4)dorsalupper body surface of worms
5)ventrallower body surface of worms
6)ectodermoutermost cell layer of worms
7)mesodermmiddle cell layer of worms
8)endoderminnermost cell layer of worms
9)acoelomatesolid body in flatworms
10)coelombody cavity
11)gastrovasculargut with a single opening in flatworms
12)absorptionhow flatworms exchanges gases with their environment
13)anteriorhead or front end of a flatworm
14)cephalizationanterior end with sensory structures concentrated there
15)parasiteworms that live on or inside the body of their host
16)turbellariaclass of flatworms containing the freshwater planarian
17)pharynxextendable feeding tube on the underside of planarians
18)mucussecreted by planarians to glide across
19)flameexcretory cells in worms that filter wastes
20)gangliaclusters of nerve cells in worms
21)hermaphroditesworms that make both eggs & sperm
22)trematodaclass of flatworms containing flukes
23)suckersfound on the ends of flukes to attach to hosts
24)tegumenttough outer covering on flukes
25)cestodaclass containing tapeworms
26)scolexanterior end of a tapeworm with hooks & suckers
27)proglottidsreproductive sections of a tapeworm’s body
28)nematodaphylum for roundworms
29)tapershape of both ends of a roundworms body
30)cuticleprotective noncellular layer of roundworms
31)ascarisroundworm found in pig & horse intestines
32)hookwormsintestinal parasitic roundworm with cutting plates in it’s mouth
33)trichinosisdisease that result from eating infected undercooked pork
34)pinwormsmost common parasitic roundworm of children in the U.S.
35)rotiferaphylum of worms with a crown of cilia surrounding the mouth
36)mastaxmuscular organ of rotifers to break down food
37)pseudocoelomatesrotifers & nematodes are examples

 

Quiz Bacteria

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Name: 

Bacteria

 

 

True/False
Indicate whether the sentence or statement is true or false.
1.
Gram-negative bacteria have a thick layer of peptidoglycan that stains purple.
2.
Bacteria lack nuclei and therefore also lack genetic material.
3.
Bacterial cells have membrane-bound organelles and chromosomes.
4.
Bacterial cells are usually much larger than eukaryotic cells.
5.
Bacteria are incapable of movement themselves; they can only get to new locations by growing toward them or by forming endospores and being carried in air or water.
6.
Some bacteria cannot survive in the presence of oxygen.
7.
When bacteria undergo nonreproductive genetic recombination, their bacterial chromosome is altered.
8.
Certain antibiotics have become ineffective against certain strains of bacteria. These bacteria have developed a resistance, which may be passed on from one generation of bacteria to the next.
9.
The photoautotrophic bacteria are the only bacteria that are indirectly beneficial to humans.
 

Completion
Complete each sentence or statement.
10.
Spiral bacteria are called ____________________.

11.
Spherical bacteria are called ____________________.

12.
Rod-shaped bacteria are called ____________________.

13.
The procedure used to distinguish between two types of bacterial cell wall structures is called ____________________.

14.
Protective structures that some bacteria may form under harsh conditions are ____________________.

15.
The cell walls of Gram-negative eubacteria are composed of a combination of polysaccharide and polypeptide called ____________________.

16.
Bacteria that obtain their energy by removing electrons from inorganic molecules, rather than obtaining energy from the sun, are called ____________________ bacteria.

17.
In general, organisms that obtain their energy from sunlight are called ____________________.

18.
Bacteria that are heterotrophic and feed on dead organic matter are called ____________________.

19.
A(n) ____________________ is a substance that can be obtained from bacteria or fungi and can be used as a drug to fight pathogenic bacteria.

20.
Many bacteria are ____________________ and play an important role in recycling carbon, nitrogen, and other elements, while other bacteria are ____________________ and assemble organic compounds from carbon dioxide, nitrogen, and other elements.

 

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Quiz Viruses

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Name: 

Viruses

 

 

True/False
Indicate whether the sentence or statement is true or false.
1.
Although viruses do not consist of cells, biologists consider them to be living because they are capable of reproduction.
2.
Wendell Stanley made the important discovery that viruses are not cellular.
3.
Viruses consist of RNA or DNA surrounded by a coat of protein.
4.
Prions are the smallest known particles that are able to replicate.
5.
A virus can only reproduce by controlling a cell.
6.
People can contract the influenza virus more than once because the virus tends to mutate rapidly, avoiding the actions of the immune system.
7.
Smallpox is caused by bacteria.
8.
Chickenpox and shingles are caused by the same virus.
9.
The viruses that have been linked to human cancers are usually transmitted through the air.
 

Completion
Complete each sentence or statement.
10.
The protein coat of a virus is called a(n) ____________________.

11.
Most viruses occur in the shape of a(n) ____________________ or a(n) ____________________.

12.
A virus that transcribes DNA from an RNA template is called a(n) ____________________.

13.
An enzyme called ____________________ manufactures DNA that is complementary to a virus’s RNA.

14.
____________________ are bacterial viruses with a polyhedral head and a helical tail.

15.
All viruses reproduce by taking over the reproductive machinery of a ____________________.

16.
Viruses that infect a host cell and have their nucleic acid replicated but do not harm the host cell are in a ____________________ cycle.

17.
A viral DNA molecule formed from an RNA virus is called a(n) ____________________.

18.
The virus that causes AIDS is called ____________________.

19.
Some viruses are thought to induce ____________________, a disease characterized by uncontrolled cell division.

20.
An example of an emerging virus is the  ____________________ virus.

 

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