Bacterial Strain Culture Media And Plasmid DNA Biology Essay

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Two important areas in microbial genetics- plasmid extraction and transformation will be explored. Plasmid extraction is a crucial procedure to prepare purified plasmid DNA. It ensures the removal of contaminants in a bacterial cell lysate which includes nucleic acids, both genomic DNA and RNA, proteins and lipids, all of which will affect the transformation process of the bacterial cells. In this practical, pUC18 plasmid was employed as the cloning vector to carry the gene of interest, ampR into the bacterial host cells. This artificial plasmid has been genetically engineered to contain ampR which encodes the protein B- lactamase that will degrade ampicillin, thereby conferring antiobiotic resistance to the bacterium that possess this plasmid. The purpose of transformation is to introduce this pUC18 plasmid into the competent E.coli cells. As ampicillin acts as a selective pressure, only cells that have successfully acquired the plasmid would be able to grow on the ampicillin medium, and formed observable colonies. In this practical, we have drawn up an altered protocol that was carried out together with the standard one in order to examine the effect of the alteration. Our hypothesis was there will be a reduction of plasmid DNA (pUC18) that are transformed into the competent E.coli cells, hence transformation efficiency will be significantly reduced. 

2. Materials and Methods

Bacterial Strain, culture media and plasmid DNA, competent cells used

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Escherichia coli (pUC18) strain, Luria-Bertani (LB) medium and LB + ampicillin medium, 5 μl and 15μl plasmid DNA, 1ml competent E.coli cells.

Buffers used and its functions

Buffer PD: Resuspension Functions

50 mM Tris-HCl pH 8.0 Maintenance of pH

10 mM EDTA Indirect inactivation of nuclease activity

10 μg/ml RnaseA Degrades RNA

Buffer PD2: Lysis

circular plasmid DNA 200 mM NaOH Denature proteins and chromosomal DNA (not the closed

are components of the cell membrane1% SDS (w/v) Causes cell lysis by breaking down fatty acids and proteins which

Buffer PD3:Neutralisation

guanidinium hydrochloride Denature proteins

plasmid DNA acetic acid Precipitate chromosomal DNA, separating it from

W1Buffer: Wash

binding of DNA to the silica membrane

membrane

guanidine hydrochloride Denature residual proteins as well as facilitate the

isopropanol Prevent DNA loss during washing step

silica, so that plasmid will be easily eluted.

Wash Buffer (70% ethanol) Removes salt causing plasmid to be less tightly bound to

In this practical, extraction of plasmid DNA was carried out using the High-Speed Plasmid Mini Kit which allows plasmid DNA to be quickly isolated from the bacterial cultures. The purified extracted plasmid DNA were then used for transformation of the competent E.coli cells. To the first two tubes each containing 300μl of the competent cells, 5 and 15μl of the purified plasmid DNA were added respectively and 10 μl of water was added to the third tube containing the competent cells. Next, these tubes were left on ice for 30mins to allow the adhering of the plasmid DNA to the cells. Then heat shock at optimum temperature of 42°C for 90secs.The cells were then plated on LB and LB + ampicillin agar plates and incubated at 37°C.

3. Results

The transformation results for both the standard and altered protocol are tabulated below.

Table 1: Transformation results for standard protocol

Volume of DNA

Concentration

Amount of plasmid DNA

No. of bacterial colonies

Plates

Rank

5 μl

Neat

117 ng

550

LB + ampicillin

3

Dilute

11.7ng

55

LB + ampicillin

5

15 μl

Neat

352 ng

1000

LB + ampicillin

2

Dilute

35.2 ng

100

LB + ampicillin

4

Negative control

(10 μl water)

Neat

-

LB only

1

Neat

-

-

LB + ampicillin

6

Table 2: Transformation results for altered protocol

Volume of DNA

Concentration

Amount of plasmid DNA

No. of bacterial colonies

Plates

Rank

Based on relative density

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5 μl

Neat

110 ng

250

LB + ampicillin

3

Dilute(10-1)

11.0ng

25

LB + ampicillin

5

15 μl

Neat

330 ng

550

LB + ampicillin

2

Dilute(10-1)

33.0 ng

55

LB + ampicillin

4

Negative control

(10 μl water)

Neat

-

LB only

1

Neat

-

-

LB + ampicillin

6

The concentration for the standard protocol measured using NanoDrop spectrophotometer was 70.4ng/μl. Sample calculation of the plasmid DNA concentration for standard protocol is as follow:

Amt of plasmid DNA in 5 μl: 70.4 X 5= 352 ng

352ng of plasmid DNA will be transformed into 300 μl of competent E.coli cells, however, since only 100 μl of cells were plated,

For 5 μl

Neat suspension: 352/ 3 = 117 ng of plasmid DNA will be present

10-1 diluted suspension: 352/ (3X10) = 11.7ng of plasmid DNA will be present

Following the above calculation,

For 15 μl:

Neat suspension: 1056/3 = 352 ng of plasmid DNA will be present

10-1 diluted suspension: 1056/ (3X10) =35.2 ng of plasmid DNA will be present

Rankings on the relative colony density

Greatest number of bacterial colonies were observed for the negative control LB plate so it was ranked No.1. This is because LB medium is a nutrient growth substrate which means that it will not preferentially grow one kind of bacteria over another. Since the negative control contains only water and competent bacterial cells, hence profuse growth of bacteria will take place on the LB plate. Therefore much denser colonies were observed as compared to the rest of the LB + ampicillin plates which preferentially grow ampicilin- resistant cells. The negative control was the only one which uses LB plate so as to act as an indicator of the viability of the bacterial cells after being subjected to the heat shock process. If no colonies were formed after incubation, there may be high possibility of the cells getting killed from the heat shock process, due to an exceed in time limit or temperature.

No colonies were observed for the negative control LB + ampicillin plate as no plasmid DNA was introduced to the competent cells As a result, no transformation would have taken place that would confer resistance to the bacterial cells, thus no growth on the ampicilin medium, so it was ranked last.

15μl of the neat sample was ranked 2nd highest in colony density followed by the 5μl neat sample. This is due to the higher amount of plasmid DNA present in the more concentrated neat sample, leading to higher transformation efficiency. Therefore more bacterial cells will be conferred ampicillin-resistant property and thus able to grow on the selective ampicillin medium.

The 15μl diluted samples was ranked 4th, and the 5μl diluted sample ranked 5th.This is due to a significant decrease in the amount of plasmid DNA present. The altered protocol follows the same ranking.

Observations for standard and altered protocol

With reference to Table 1 and 2, the number of colonies observed for the standard protocol was about twice as much as those in the altered one, therefore a much higher colony density was observed. For example, 5 μl of neat sample for standard shows about 550 colonies while the altered had about 250 bacterial colonies.

However, the size of the bacterial colonies for both standard and altered was quite small. This may be due to insufficient incubation time.

Table 3: Concentration and purity of extracted purified plasmid DNA

Standard protocol

Altered protocol

Concentration= 70.4ng/ μl

(A260/A280) = 1.87

(A260/A230)= 1.87

Concentration= 66.0ng/ μl

(A260/A280) = 1.87

(A260/A230)= 1.87

The concentration and purity of the extracted plasmid DNA preparation (pUC18) were measured using the NanoDrop spectrophotometer. The plasmid DNA sample purity grade was estimated using the 260/280 intensity band ratio. Samples with ratio between 1.8 - 2.0 are considered to be of good purity, with little or no contaminants. Lower ratios would otherwise indicate presence of contaminants like proteins, phenol which absorb strongly at or near 280nm. The secondary measure for DNA purity will be the 260/230 intensity band ratio, which indicates the level of salt carryover in the DNA sample. A good preparation will have a ratio of more than 1.8. Lower ratios again will indicate presence of contaminants which absorb strongly at or near 280nm.

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With reference to Table 3, a good concentration and purity were obtained for both standard and altered protocol. The ratio fell within the range of a good sample purity grade. Hence indicating the plasmid DNA samples to be relatively free of contaminants. This is very important as the presence of contaminants may disrupt the transformation process and greatly lower transformation efficiency. Transformation efficiency is a measure of the number of E.coli cells that have successfully uptaken the plasmid DNA (pUC18). It represents the number of colony-forming units, divided by the 1 mg of DNA. Therefore any difference in the number of colonies obtained when comparing the standard and altered protocol would largely be due to the alteration- which was the change in temperature in step 5 of practical 3 rather than other factors like low purity or concentration of the plasmid DNA used.

4. Discussion

The intent of the altered protocol was to examine the effects of removing heat shock process, (and in place leaving the mixture of competent cells and plasmid DNA at room temperature) on transformation efficiency. The different results obtained for the standard and altered protocol will be interpretated here.

Bacteria cells used were made competent artificially to take up plasmid DNA by suspending in a solution of CaCl2 solution. The calcium ions will reduce repulsion resulting from the like- charges of the negatively-charged plasmid DNA and that of the lipids, allowing the entry of the DNA. Heat shock at 42 °C however, serves to facilitate this process even further by activating heat shock proteins (HSPs) which causes formation of pores on the bacterial membrane. Thermal gradient was established, so the plasmid DNA will move from a higher temperature outside the cell to a lower temperature inside the cell. Once inside, during the log phase of growth, bacteria will start transcribing the genes found on the pUC18 plasmid, one of them being ampR. Protein B- lactamase is secreted into the surrounding medium, degrading ampicillin. Hence successful transformants will be able to grow on the ampicillin forming observable colonies while nontransformed cells will be killed by the antibiotic and not grow. In this way, ampicillin medium acts as an antibiotic selection for the transformants.

The number of colonies observed for the standard protocol was about twice as much as those in the altered one. This is because in our altered protocol, bacterial cells were left at room temperature instead of the standard heat shock treatment of 42°C. HSPs will not be activated, and pores formation will significantly reduce. Also, thermal gradient established was much diminished. Hence there was a great reduction in the rate at which the plasmid DNA would have been able to enter the cells through the pores, reducing transformation efficiency. So fewer colonies of the transformed E. coli cells was observed growing on the LB + ampicillin agar plates (altered)

5. Conclusion

The results obtained supports our hypothesis that without heat shock, transformation efficiency would be significantly reduced, hence the study of this practical shows the heat shock step at 42°C , the optimum temperature for DNA uptake process , is important for the enhancement of transformation efficiency.