Transformation Of Ecoli Cells By Green Fluorescent Protein Biology Essay

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The experiment was designed to clone the EGFP sequence into a plasmid containing histidine tag by PCR amplifying the EGFP sequence. For the pBluescript plasmid to accept the amplified gene it was cut with EcoRI restriction enzyme and was subjected to various chemicals. The EGFP sequence was cloned into the multiple cloning site of the pBluescript plasmid. The plasmid with the PCR product ligated into it was then transformed into bacteria and the bacteria was then placed onto the agar plates with ampicillin in order to select for bacteria with inserts. Several colonies were formed onto the agar plates and the bacteria that had been successfully transformed was selected and expanded in culture. Plasmid were extracted from bacteria and analyzed by restriction digest to establish if they had PCR-insert. The gel photo indicated no bands and absence of PCR- insert.

INTRODUCTION

Gene cloning is a technique wherein the DNA can be manipulated externally and can then be returned back to the organism in order for it to function normally (Lodge, 2007). The importance of this technique is that it can provide a pure sample of an individual gene, separated from all other genes in the cell.

The method involves cloning a piece of DNA obtained from an organism into a host such as Ecoli. The bacterium Ecoli is then allowed to produce colonies. Cells that carry the copies of the plasmid will produce colonies and the bacteria in which the plasmid is absent will be killed by the antibiotic as a result, will not produce any colonies. Using these technology plasmids containing the gene of interest can be produced and then it can be introduced into cultured cells which reproduce and replicate the DNA. The expression of DNA can lead to desirable genes, yielding a desirable protein which can then be produced in large quantities (Brown, 2006).

This experiment involved cloning of enhanced green fluorescent protein (EGFP) contained within the template DNA into a pBluescript plasmid containing a histidine tag. The pBluescript plasmid contains an ampicillin resistance gene and a multiple cloning site (MCS) integrated in the lacZ gene. The EGFP was cloned into the MCS by cleaving the plasmid with the EcoRI restriction enzyme. It cuts the DNA producing a construct in which the EGFP protein and his-tag peptide are in the same frame. Sticky ends are formed which are then made permanent by DNA ligase.

The pBluescript plasmid used in this experiment had two genes namely ampR gene which codes for a protein that makes the cell containing this gene resistant to the antibiotic ampicillin and lacZ gene forms a functional β- galactosidase enzyme which breaksdown lactose into glucose and galactose. It also catalyzes Xgal (5-bromo-chloro-3-indolyl-β-D-galactoside), an artificial substrate into blue products. The EGFP gene was inserted into the lacZ gene which disrupted the lacZ protein thereby stopping the activity of β-galactosidase. As a result, lac- strains produced white colonies as they were unable to produce Xgal and lac+ strains produced blue colonies as they were able to process Xgal into 5-bromo-4-chloro-indoxyl (Lodge, 2007).

The aim of the experiment was to plan and execute a gene cloning experiment by cloning the EGFP sequence into a plasmid containing histidine tag.

MATERIALS AND METHODS

TO CARRY OUT THE RESTRICTION DIGEST OF THE PLASMID

20µl of the pBluescript plasmid was digested with EcoRI restriction enzyme. Following components were pipetted into the tube which was then digested at 37°C (for 1hr-overnight)

AMOUNT

COMPONENTS

2µl

10X restriction enzyme

6.13µl

DNA

1µl

Restriction enzyme (EcoRI)

10.87µl

H2O

TO PREPARE PCR PRODUCT

Three PCR tubes were prepared by adding the following components.

AMOUNT

COMPONENTS

40µl

Template DNA(EGFP plasmid)

4µl

Forward primer

4µl

Reverse primer

48µl

Master mix

All the components were mixed and out of the total volume about 24µl were removed into each of the three PCR tubes. And the tubes were placed in the thermocycler.

TO PERFORM CONTROL LIGATIONS

All the 3 PCR samples (60µl) were collected and purified on a QIAquick column. The gel tank was set and about 2µl of the dye was added to the 10µl of the PCR product and the sample was loaded onto the gel and was observed. After sometime out of 20µl of the original digested plasmid, 2µl was kept as control. About 2µl each of the digested plasmid and uncut pBluescript plasmid was combined with 2µl of dye and 8µl of H2O and both the plasmids were loaded onto the gel next to the PCR sample along with the marker which had 1µl of DNA and some dye in it. The remaining 50µl of the PCR sample was digested with the restriction enzyme EcoRI. The 80% (16µl) of the digested plasmid was then treated with alkaline phosphatase. The restriction enzyme and the phosphatase was heat inactivated at 75°C for 15 mins. The final PCR product was then purified on a QIAquick column and the ligations were set up as follows with each tube containing the following components.

REACTION

RAPID LIGASE

BUFFER

pBluescript

VERSION

LIGASE

PCRINSERT

(Vector:Insert)

H2O

1

5µl

UNCUT- 50ng (1.25µl)

NO

NO

Upto 10µl

(3.75µl)

2

5µl

CUT- 50ng (1.25µl)

NO

NO

Upto 10µl

(3.75µl)

3

5µl

CUT- 50ng (1.25µl)

1µl

NO

Upto 10µl

(2.75µl)

4

5µl

CUT- SAP- 50ng (1.25µl)

1µl

NO

Upto 10µl

(2.75µl)

5

5µl

NO

NO

25ng (1µl)

Upto 10µl

(4µl)

6

5µl

CUT - SAP- 50ng (1.25µl)

1µl

3:1 molar

ratio

(0.1µl)

Upto 10µl

(2.65µl)

7

5µl

CUT-SAP-50ng (1.25µl)

1µl

1:1 molar

ratio

(0.3µl)

Upto 10µl

(2.45µl)

8

5µl

CUT- SAP-50ng (1.25µl)

1µl

1:3 molar

ratio

(0.9µl)

Upto 10µl

(1.85µl)

9

5µl

CUT- SAP -50ng (1.25µl)

1µl

Digested PCR product provided

(0.9µl)

Upto 10µl

(1.85µl)

TRANSFORMATION OF Ecoli WITH LIGATION MIXES

The ligations were incubated for 10 mins at room temperature and were then placed on ice. To each tube 100µl competent cells were added and the tubes were placed on ice for 15 mins. This was followed by subjecting the transformations to heat shock by placing the tube at 42°C for 2 mins. Finally 0.9ml L-broth was added to each tube and incubated at 37°C for 1 hr. after incubation 100µl of sample from each tube was spread onto ampicillin- agar plates containing X-gal and IPTG. The agar was allowed to dry and the plates were incubated. This resulted into development of blue and white colonies onto the agar plates. Two white colonies were picked up using a toothpick and were transferred to 5ml of liquid broth (containing ampicillin). The 5ml cultures were then grown overnight with shaking at 37°C.

PLASMID DNA EXTRACTION- PLASMID MINIPREPS

The 5ml Ecoli grown overnight was collected and the cells were micro centrifuged to obtain a pellet. To the pellet 250µl of buffer P1, buffer P2 and 350µl of buffer N3 was added and the contents were mixed by inverting the tube 4-6 times. The tube was then centrifuged for 10 mins and supernatant collected was applied to the QIAprep spin column and was centrifuged for 30-60sec. 0.5ml buffer PB was added to the column and centrifuged again for 30-60sec. Also, 0.75ml buffer PE was added to the column and centrifuged for another 30-60sec followed by centrifuging the column for another 1 min to remove the residual wash buffer. The QIAprep column was placed in a 1.5ml microcentrifuge tube and 50µl of buffer PB was added and the column was allowed to stand for 1min and then centrifuged for 1min.

RESTRICTION DIGESTION TO RELEASE THE INSERT

The amount of DNA in the plasmid miniprep was quantified by OD at 260nm (ie 5µl of plasmid minipreps in 95µl H2O). The digest was then setup (with BamHI and HindIII) using 1µg of each of the two minipreps as follows.

TUBE 8+ (total vol - 20µl)

TUBE 8- (total vol - 20µl)

2µl of 10Xrestriction enzyme buffer

2µl of 10X restriction enzyme buffer

1µl of H2O

3µl of H2O

1µl of BamHI restriction enzyme

No enzyme

1µl of Hind III restriction enzyme

No enzyme

15µl DNA

15µl DNA

TUBE 9+ (total vol - 20µl)

TUBE 9- (total vol - 20µl)

2µl of 10Xrestriction enzyme buffer

2µl of 10X restriction enzyme buffer

1µl of H2O

3µl of H2O

1µl of BamHI restriction enzyme

No enzyme

1µl of Hind III restriction enzyme

No enzyme

15µl DNA

15µl DNA

TUBE 5 (total vol - 20µl) Pbs+

TUBE 6 (total vol - 20µl) Pbs-

2µl of 10Xrestriction enzyme buffer

2µl of 10X restriction enzyme buffer

14µl of H2O

16µl of H2O

1µl of BamHI restriction enzyme

No enzyme

1µl of Hind III restriction enzyme

No enzyme

2µl DNA

2µl DNA

1µg of each plasmid miniprep with no enzyme (tube 8- and tube 9- ) was incubated overnight at 37°C and was then digested overnight at 37°C

(+) sign indicates the presence of restriction enzymes BamHI and HindIII.

(-) sign indicates absence of both the restriction enzymes.

RUNNING THE SAMPLES ON THE AGAROSE GEL

The gel tank was set and 10µl of the marker was loaded onto the gel. To the 20µl of all the above six samples 5µl of dye was added and in all 15µl of each of the six samples was loaded onto the gel. The gel photo was taken and visualized.

RESULTS

TO RUN THE PCR SAMPLE ONTO THE GEL BEFORE PERFOMRING THE LIGATIONS

The gel tank was setup and the marker was loaded onto it. This was followed by addition of 10µl of purified PCR product, about 12µl each of plasmid digested with EcoRI restriction enzyme and the uncut pBluescript plasmid. The gel photo obtained showed one thick bright band for the uncut plasmid,two thick, bright bands for the plasmid digested with EcoRI and one band for the PCR sample (gel photo 1).

TRANSFORMATION OF Ecoli WITH LIGATION MIXES

After incubation the agar plate containing X-gal and IPTG onto which 100µl of the sample from each tube was spread was observed. The number of blue and white colonies on each plate was counted manually and recorded.

PLATES

NO OF BLUE COLONIES

NO OF WHITE COLONIES

1

1244

6

2

2932

46

3

2612

43

4

2872

46

5

0

0

6

876

27

7

2476

37

8

1300

33

9

1696

20

TO TRANSFORM CONTROL LIGATIONS

Following table illustrates the predictions for the first five control ligations carried out and the results that were observed along with the ideal observation that should have been.

REACTIONS

PREDICTIONS

REASON

OBSERVED RESULTS

1

Yes

The plasmid is circular and the transformation reaction works.The plasmid generates resistance to the antibiotic ampicillin in the bacteria.

Lawn of bacteria

2

No

As the plasmid was digested with the restriction enzyme it became linear and as soon as it enters into the bacteria the linear DNA is degraded

Lawn of bacteria

3

Yes

Because the ligase cause the plasmid to recircularise and there is no insert

Lawn of bacteria

4

No

As the plasmid was phosphatased, it cannot recircularise even on addition of ligase. Hence, the linear plasmid would be degraded once it gets into the bacteria.

Several colonies of bacteria

5

No

As there is only PCR sample present and no plasmid

No colonies

RESTRICTION DIGEST TO RELEASE THE INSERT

The amount of DNA in the plasmid miniprep was quantified by measuring its absorbance using a UV/Vis spectrophotometer (Du@730 Beckmann coulter). The absorbance readings were taken at wavelength of 260nm and 280nm for the tubes 8 and 9 which contained individual white colony collected from the ampicillin agar plates containing Xgal and IPTG respectively. Following readings were obtained.

miniprep

Æ› 260nm

Æ› 280nm

Ratio

Tube 8

0.050

0.033

1.522

Tube 9

0.015

0.011

1.366

RUNNING THE SAMPLES ON THE AGAROSE GEL

The gel tank was set up and all the six samples namely tubes 8+, 8-, 9+, 9-, 5 and 6 were loaded onto the gel and the gel photo was obtained (gel photo 2). The gel photo was over exposed and no bands were visible on the gel photo indicating absence of insert.

DISCUSSION

TO RUN THE PCR SAMPLE ONTO THE GEL BEFORE PREPARING THE LIGATIONS

The PCR sample was first purified to remove unused primers, primer-dimer, unincorporated dNTPs and Taq polymerase. A small portion of the sample was then loaded onto the gel to check if it worked. Along with the PCR sample the plasmid cut with EcoRI restriction enzyme and the uncut pBluescrit plasmid was also loaded onto the gel. Lane 1 had one bright and thick band of 2kb as the plasmid was uncut. Lane 2 had plasmid cut with restriction enzyme EcoRI. As a result two bands of 5kb and 2kb were visible due to a cut introduced in the strands of the DNA by restriction endonuclease. The lighter and the slow moving band is the relaxed form (single stranded nick) and the brighter, faster moving band is the supercoiled form of the plasmid DNA. In lane 3 the PCR sample gave only one brighter band of about 0.5kb (124ng). This data was then used to calculate the PCR insert (vector: insert ratio) for reaction 5 to set up the ligation reaction.

TO TRANSFORM CONTROL LIGATIONS

For reaction 2, lawns of bacterial colonies were visible. The results obtained were not same to what was predicted. As the plasmid was digested with restriction enzyme it would have been a linear plasmid. When it enters the bacteria the linear DNA is degraded resulting in no colonies. However the formation of colonies indicated that the EcoRI restriction enzyme was unable to cut the plasmid efficiently. For reaction 4, several colonies of bacteria were visible. The plasmid was treated with alkaline phosphatase before subjecting it to ligation. The phosphatase catalyses the removal of the 5' phosphate groups from the digested pBluescript thereby preventing religation of the plasmids. As a result, the linear plasmid is degraded once it gets into the bacteria resulting in no colonies. The results indicated that the phosphatised failed and the plasmid still had phosphate groups. The ligase in turn recircularised it leading to colonies.

RUNNING THE SAMPLES ON THE AGAROSE GEL

The gel photo was overexposed as a result no bands were obtained. This might be due to excess amount of sample being loaded onto the gel or may be some error. One overexposed spots were visible for samples 8+ and 9+ which had both the restriction enzymes, BamHI and HindIII. The distance travelled by these two spots were the same as that travelled by the sample Pbs+ containing the same restriction enzymes. The sizes of the bands were compared to that of the DNA ladder and were found to be about 3kb (120ng). For the samples 8- and 9- which had no restriction enzyme also travelled the same distance as that of the sample Pbs- . The sizes of the bands were 2kb (140ng). As the distance travelled by the sample is pBluescript. Also, appearance of no bands indicated that the sample had no insert.

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