Conjugation Lab Report BIOL6B PDF

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Conjugation Lab Report BIOL6B...

Description

Conjugation Process of Conjugation Conjugation is one of three methods for gene transfer within bacteria. It is the transfer of a small copy of DNA, called the plasmid, from one cell to another: Typically occurring between eukaryotic cells. All bacteria have chromosomes, however, some bacteria have evolved to have an extra chromosomal DNA called a plasmid, which allow them to carry genes that allow bacteria to grow in new environments. In order for conjugation to occur, a plasmid donor cell and plasmid recipient cell must be present. Protein structures called pilli pull the cells together, where a copy of the donor cell is transferred to the recipient cell. The end result is that the recipient gains a new plasmid, with the donor still retaining theirs. Antibiotics and antibiotic resistance Antibiotics are chemicals that kill bacteria or prevent them from growing. In this lab, Ampicillin (Amp) has a resistance gene in a form of a plasmid which inhibits bacterial enzyme transpeptidase needed for cell wall synthesis, eventually causing the cell to be lysed due to the faulty cell wall. Streptomycin (Strep) resistance gene is directly on the chromosome; it inhibits bacterial protein synthesis by binding to a component of the ribosome, destroying it which kills the bacteria. Antibiotic resistance genes are often carried on plasmids, which can be copied from cell to cell by conjugation. Biological Significance of Conjugation Bacteria reproduce asexually through binary fission, and undergo horizontal gene transfer with each bacterial “offspring” being genetically identical to the parent. This has a large consequence on genetic diversity, as if a pathogen were to infect one cell: the whole population would be wiped out. By conjugating, bacteria are able to withstand pathogenic and antibiotic factors: allowing them to survive. Furthermore, this holds a significant evolutionary difference on bacteria because plasmids allow for efficient and quick transfer of DNA. For instance, Escherichia coli bacteria (e.coli) that are Strep resistant, carry their resistance located directly on their chromosomal DNA. Although it is still possible to transfer this DNA to a non-Strep resistant cell: it takes more resources, is less efficient and is much larger to transfer in contrast to a plasmid. Goals of Experiment and Predictions In this experiment we will examine if our bacteria have conjugated by examining quantitative plates, and test for conjugation efficiency by examining qualitative plates. We predict that the bacteria which has conjugated will survive all plates, whereas the bacteria which only have Amp and Strep strain will be able to survive their respective plates. Bacteria without any antibiotic resistance will only grow on plates without antibiotics. Methods

CI

C II

Predictions: The streaked plates will show the conjugation of the CI & CII strains of E. coli since they will be plated on different plates with antibiotics. The CI+CII parts of the qualitative test will all appear

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Both C I & C II Add 0.5ml of each

CI Add 1ml

C II Add 1 ml

Qualitative

Incubate for 20 no antibiotics

C I

C II

C II

CI + CII

CI + CII

strep

amp

C I

C I

C II

CI + CII

After

C II

CI + CII

20 minutes of incubation, use streak cells from CI, CII and CI & CII onto the respective sections on plates above. Pattern is the same for all plates (in terms of CI, CII, and combo locations). Quantitative

amp + strep

10^-1

no antibiot

10^-3

10^-5

amp + strep

10^-1

10^-3

Results:

10^-5

Table 1: Initial Conjugation (Qualitative Plates)

We put 100µl on each plate and spread them with an inoculating loop.

Description: Table 1 exhibits whether growth, in the form of colonies, was present or not. e.coli Strain

No Antibiotics

Amp

Strep

Amp + Strep

cI

(+)

(-)

(+)

(-)

cII

(+)

(+)

(-)

(-)

cI + cII

(+)

(+)

(+)

(+)

Legend cI: Streptomycin Resistant Strain of e.coli

Amp: Ampicillin is present

cII: Ampicillin Resistant Strain of e.coli

Strep: Streptomycin is present

cI + cII: Conjugated Strain of cI and cII

Amp+Strep: Both Ampicillin and Streptomycin are present

(-): No Growth

(+): Growth

Table 2: Conjugation Efficiency (Quantitative Plates) Table 2 shows quantitative data, the number of colonies on our no antibiotics and Amp+Strep plates, collected for efficiency of DNA conjugation.

10-1x dilution

10-3x dilution

10-5x dilution

No antibiotics

TMTC

TMTC

86 Colonies

Amp + Strep

69 Colonies

N/G

N/G

Legend TMTC: Too Much/Many To Count

N/G: No Growth

Discussion The experiment demonstrated that antibiotics resistance was transferred from one cell to another due to the results of successful conjugation. Two strains were used in this experiment: Escherichia coli with Strep resistance and Escherichia coli with Amp resistance. Alone, they would be able to survive in their specialized antibiotic environment but would fail to survive if either strain was placed on a plate with both Amp and Strep resistance. However, when using conjugation techniques on both strands, we found that the Escherichia coli were able to survive our LB+Amp+Strep plate. To ensure that our bacteria did not already have Amp+Strep resistance, we streaked the two strains on separated areas on the LB+Amp+Strep plate and found no growth. The other experiments that we might do to be more sure of what is happening at the DNA level is running another plated samples with no antibiotics, Amp, Strep and Amp+ Strep to make sure again the DNA transformation happened successfully. Referring to Table 1: Initial Conjugation to see no antibiotics showed positive growth for CI, CII and CI+CII to ensure that growth has incurred. The LB amp plate had shown no growth for CI and growth for CII and CI+CII (growth of conjugated CI). Then LB strep plate showed no growth for CII and growth for CI and CI+CII (growth for CI and conjugated CI). Finally the LB amp+strep that had no growth for CI and CII but growth for CI+CII which the conjugated CI has grown. Knowing these plates proved valid reasons on why there would be growth in these Escherichia coli strains. The overall results proved proved that the E.Coli strains grew where they were supposed to and that the DNA from CII was transferred to CI. Although conjugation did occur, the process was not very efficient. Our calculations are as follows: The CFU/ml in the conversion of CFU/μL times 1000μL/mL times (CFU/mL)/dilution factor. From our results the calculations, gives us 86,000,000CFU/mL for no antibiotics while Amp+strep was at 6,900CFU/mL. Then we need to calculate the our conjugation efficiency by dividing the Amp + strep by no antibiotics which resulted 8.02x10-5. For the quantitative part of the lab we used the no-antibiotics plate and Amp+Strep plate with the dilutions of 10-1, 10-3 and 10-5. As seen in our results, our no-antibiotics 10-1 and 10-3 plates had a lawn of growth: causing us to be unable to count the number of colonies. However, our 10-5 plate had 86 colony forming units (CFU). Our Amp+Strep 10-1 plate had 69 colony forming units. The rest of the dilutions for Amp+Strep showed none which made sense because the amount of colonies we started with was very little. The Conjugation Efficiency as a whole for the class was very low, due to conjugation not being an efficient form of gene transfer as it needs 2 compatible mating types, contact via pillus and the successful transfer of a plasmid copy from one bacteria to another. Conclusion: The overall condition of the Conjugation lab was successful because transformation has

occurred, conjugation occurred, calculating the CFU/mL and conjugation efficiency were precise to the classroom data. Everything during the conjugation lab occurred like it was expected to, except for the minor contamination error regarding the LB+amp plate. Another way to look deeper into the conjugated bacteria is to take the cells, cut them and run the DNA through an agarose gel electrophoresis....