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Laboratory Report on Gene Cloning into Expression Vector

Abstract

Gene cloning involves manipulation of a given cellular component to isolate the desired gene

properties. Isolation of particular cellular component forms the core of the cell chemistry and

biology. For a gene to be efficiently cloned, it must be inculcated in a larger medium that will

allow sufficient expression of its markers. The vectors form the structures viable enough to

encourage the manipulation of the genes to get the desired efficacy. Mostly used vectors are the

plasmid vectors. They are ideal because of their neutrality nature and the growth favoring

characteristics. The cloning vector obtained from the plasmid is useful and provides a good

binding for the foreign DNA fragments which allows for the elucidation of cloned gene. This

laboratory report explores on how gene cloning is altered into an expression vector. It also

highlights on the Polymerase Chain Reaction and how the process is used to amplify the cloned

genes into the desired particles. The last section explains the findings of the experiment.

Keywords: cloning vectors

Polymerase Chain Reaction

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Introduction

For any desired effect of gene cloning to have an impact, there must be a proper layout on

the ways to elucidate the genes properly. The use of expression vector is effective method since

the vectors provide the necessary requirements and conducive media for an insertion of foreign

DNA genes. In the experiment, tomato cells were used as the expression vector to plant with the

gene of interest. Ideally, for a successful experiment to be conducted on the gene cloning,

different sets of parameters must be taken into consideration. First, the gene has to be isolated.

Secondly, the gene has to be cloned into a vector for a greater expression, and lastly, the gene

must be expressed and translated to give the desired aims of the experiment (Brown 10). The

laboratory experiment conducted followed the same fashion. Adjustments were made in the

vector to meet the typical qualities of eukaryotic cells. The first step taken into consideration was

the use of promoter sections, Kozak sequence and inserting appropriate start and stop codons. In

addition to the requirements, kanamycin resistance gene was used as selected marker to identify

the cells that have taken up the DNA.

Materials and Reagents Used

Isolated cDNA gene

Plasmid, pXCN

Escherichia coli strain

5microlitres buffer

3microlitres MgCl2

1microlitre of dATP

40microlitres purified PCR Product

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1microliutre Taq DNA polymerase

Incubator

Ice cubes

Test tubes

Method

The above reagents were added in a sequential manner while observing the time intervals to

realize the efficiently purified product.

One purification was done, transformation protocol followed.

The competent cells were left for some time to thaw on the ice.

5microlitres of litigation mix was then added with a lot of caution to the competent cells and the

mixture left for further 10 minutes on the ice.

The tubes were then put into a floating tube holder and placed in a 42 degrees Celsius water bath

for 1 minute to achieve acclimatization temperatures.

The cells were immediately put back on ice and kept for further 2 minutes

After that, 1milliltre of LB was introduced to the mixture and subjected to incubation for 60

minutes at 37 degrees Celsius.

On completion of the incubation, the cells were spun to concentrate them on LB plus kanamycin.
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Week 2

Screening the Plasmids

This part of the experiment was mainly aimed at screening the vector to identify if the

DNA fragments are correctly inserted as desired. The technique used was Polymerase Chain

Reaction. The bacteria colonies formed were screened to make sure that the plasmids were in

line with the size of wanted genes and the standard ranges.

Method

Each bench set up a 4times PCR master mix. The required master mix estimated for the

experiments was 100 microliters.

The master mix used was kept on the ice to maintain the ambient temperature.

The ingredients were added in the following proportions; 2.5ul and 25ul of 10x buffer

were added to 1x volume and 4xMaster mix of 25ul and 100ul respectively. Magnesium

Chloride was added to the same mixtures starting from a volume of 0.75ul and 3ul. Primer mix

was then added to the same mixture starting with 0.5ul and 2ul to the different master mix

volumes. dNTPs were added in the same pattern but in different volumes of 0.5 and 2ul. Water

ice then followed the addition sequence in the order of 20.15ul and 80.6ul. Lastly, Taq

Polymerase was added starting with a volume of 0.125ul and 0.5ul to the respective master mix

volumes.

After the additions, the solutions were gently mixed using 80ul set pipette avoiding

bubbles.

25ul of the solution was then transferred into the PCR tube and the DNA template from

part of the bacterial colony taken. The samples; were then set to run in PCR machine using
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standard conditions. The denaturation, annealing and elongation temperatures were set as 94, 58

and 72 degrees Celsius respectively with following timings per cycles made.

After the PCR run, the product was loaded onto the agarose gel and results analyzed.

Results

PCR Screen 1

Clear bands formed

Proper orientation of the base pairs

Clear visibility of PCR wells

Diagram

PCR Screen 1

PCR Screen 2

No clear bands on the formation of the required primers. 1000kb primer is not expressed in the

sample loaded in the gel agarose. Additionally, there is an irregular arrangement of the base

pairs.
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Diagram

PCR Screen 2

Restriction Digest Screen

There is obstruction of the primers hence no clear bands are observed. The base pairs are

irregular placed. The bands formed do not coincide with the required base pair primer expected

in the screening results.

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Diagram

Restriction Digest Screen

Blast Analysis

Blast X was used to find the nucleotide query sequence against protein database sequence. The

nucleotide sequence used covered the entire mRNA with proper alignment of the base pairs to

form stop codon, TGA.

Electrophoretogram

It is a tool used in analyzing the presence of nucleotide sequences by passing it through electric

current. The number of wavelengths depicts the presence of nucleotide sequence. In the

experiment, cytosine expressed greater wavelength followed by Adenine and subsequent base

pairs. The pattern displayed movement of the charged particles in the agarose gel.

Discussion

The PCR Screen 1 was most efficient among the other experimental methods used. It

presented a clear outline of the band coinciding with the required primer one. The insertion of

the plasmid evidenced by the presence of clear band indicates that there is the proper

introduction of the forward primer and reverse primer having points of origin. In restriction
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digest screen, the presence of BamHI restriction enzyme although tend to be valuable in

promoting the insertion of the primers into the cell, the exact area of cutting cannot be estimated

therefore raises a discrepancy on the formation of the actual band (Davis 12). When BamHI cuts

the cell, it creates a surface for appropriate insertion of both the reverse and forward primers. The

method is not effective because the precise cutting point is not known.

PCR Screen 2 defines how the recombinant plasmid orientation is formed. The band

formed is not clear, a likely explanation on the random combination of annealed forward and

reverse primers. A clear outline is thus not shown in the method. The orientation of the gene is

not inserted correctly. The sequence results of the restriction digestive show a messy situation

with irregular peaks. This is also in line with screen two results.

The Recombinant plasmid screen displays the mixed result based on the various

performances of the three screens. In the display, the variation is tied to inconsistencies realized

on the second screen and the restriction digestive screens. Therefore, no clear markings are

shown in the recombinant blot. The sizes vary and present smears instead of bright bands.

Precise and efficient gene cloning into vector expression requires a combination of

different determinants in which each plays a significant role in the insertion of the primers. A

good backup for that is the point of origin of the annealed forward and reverse primers (Unger

34). The two must face each other to form a synergy at the cutting points and create an active

formation of the plasmid.

The process of gene cloning requires the manipulation of cells to meet up the behavior

characteristics of a Eukaryotic cell. The experiment took into account the use of promoters to

enhance the binding of DNA and its expression. The cells were also manipulated to adopt a

Kozak sequence where the start and stop codons shift in the operations (Davis 20). The first
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phase of the experiment involved ligation reactions induced in Escherichia coli because it does

not have the antidote. Appropriate follow-up of the gene coding should be followed to minimize

chances of the discrepancy. In most of this case scenarios, Sanger Sequencing reactions should

be used in this cases to check where abnormal ddNTPs have been added along the sequence.

The experiment ends with the formation of cDNA library. It begins with placing mRNA in a test

tube. PolyT oligonucleotide is then mixed with the sample (mRNA) in the tube. The

oligonucleotide will bind to the 3 end of mRNA which has polyA tail. The synthesis of cDNA

library from mRNA needs an action of reverse transcriptase which alters the replication dogma

enhancing the formation of the cDNA (Unger 35). RNAse is added to eliminate the RNA strands

which will form short fragments that can later be used as primers in the formation of another

DNA strand.

Diagram of the principle of cDNA library Formation (Unger 36)

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In conclusion, gene cloning is an ongoing biomedical innovation that serves as a vital

link in the manipulation of cells to give the desired effect. The cloning of the cells to provide the

vector you want to require an excellent analysis of the orientation of forward and reverse

primers, antibody resistant gene e.g. Kanamycin and Tetracycline among others. The experiment

explicitly covered on gene cloning into an expression vector.

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Works Cited

Bio4442017 Laboratory Practical Manual

Brown, Terence A. Gene cloning and DNA analysis: an introduction. John Wiley & Sons, 2016:

10-63

Davis, Leonard. Basic methods in molecular biology. Elsevier, 2012: 7-58

Unger, Tamar"Applications of the Restriction Free (RF) cloning procedure for molecular

manipulations and protein expression." Journal of structural biology 172.1 (2010): 34-44.