M.Sc. Molecular Biology And Biotechnology Coursework
Introduction - M.Sc. Molecular Biology And Biotechnology Coursework
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Section 1:
Q1.
PCR Cyclic Condition:
PCR steps are divided in three consecutive steps and those are denaturation of the treatment strands, then annealing of the forward and the reverse primers and then extension through synthesis (W.et al 2020). This goes down in a cyclic manner to amplify the gene of interest or product. These cycles run for approximately 25 to 30 times and that sometimes vary upon the number or the amount of DNA that is provided for the yield of products. If the input is as small as 10 copies then it requires only 40 cycles. If there is no specific yield then more than 45 cycles can be allowed to apply.
Temperature cycle of PCR :
Denaturation: this is the primary step of PCR that makes the template stages for the next steps. In this step two strands separated out and in the next step primers get aneallined. In this step the temperature is always at 95 to 100 degree centigrade (Pandey et al, 2020). The hydrogen bonds are broken down by this higher heat and the range of heat is designed on the basis of the number of hydrogen bonds. Sequences that contain more GC bonds require high temperature and more time than other bonds. More the hydrogen bonds, the more temperature required.
Primer Annealing:
Two primers one forward and the other one is the backward primer annealed with the template in their specific regions and in this stage the temperature is within 50 to 68 degree centigrade. This temperature is determined by the calculation of the melting temperature. There is a general thumb rule of annealing temperature that is 3 to 5 degree centigrade than the minimum Tm of the primers. This is determined by the formula, + 2 (A+T)” (Jie, et al, 2020)and that can be more accurately calculated by “Tm + 81.5+16.6 (log[Na+]) + 0.41 (% GC)-675/ length of primer”.
PCR Components:
There are different components or reagents that involved in the PCR reaction.
DNA Template:
Template is the double helical DNA that is the gene of interest that acts as the base of reaction for amplification.
DNA polymerase:
DNA polymerase is an catalytic enzyme that acts in the PCR reaction to add the dNTPs in the optimum temperature and in optimum condition this.
Primers:
Primers are short oligonucleotide sequences that attach with the template molecules and stretched during the PCR reaction. Those are attached to the template at 50 to 65 degree C.
dNTPs:
There are 4 nucleotide molecules that are involved in the PCR reaction. Those are the deoxyribonucleotide triphosphates. These are the essential components that must needed for synthesis.
Buffer:
PCR buffers ensure that the PCR reaction is conducted under optimal conditions. The major components of PCR buffer include Tris-HCl, potassium chloride (KCl) and magnesium chloride. Tris-HCl and KCl are responsible for maintaining a stable pH during PCR. Magnesium ions act as cofactors for DNA polymerase so as to ensure proper DNA synthesis function of the polymerase during PCR. PCR buffer is usually available at 10X concentration.
Extension:
The final step of the PCR temperature cycle the Taq DNA polymerase enzyme worked for synthesis or extension of the complementary sequence. That prepares the next template for next cycle and this step is conducted within the temperature of 70 to 72 degree centigrade.
Properties of good PCR template:
The template of PCR is an extremely purified DNA that has the 30 ng to 50 ng concentration and should have the 50 to 55% of the GC content. That also needs to be free from different chemical contaminations.
Evaluation of the Primers’s Tm :
The forward primer: 5' ATCCCGGGATACCCGGGAT 3’ (19 bp)
As it is known that, a good primer should have the length within 18 to 24 bp and that 40 to 60 % of the GC content and the Tm should be within 50 to 60 degree C (Abd et al, 2020). Those primers should have the Tm within 5 degree C and should not have the regions of complementarity. In these primers the forward primer is 62 and the backward primer is 64 degree centigrade that is slightly over the expected temperature and both primers have the same Tm . the length of the primers and the GC content is as expected. As it is known that primers should not have any palindromic sequences or the “inverted repeat Sequences” that’s why the forward primer which is a palindrome sequence is not a good choice of primer.
Interpretation of the gel image:
This gel contains a 1000bp ladder and next well contains the PCR template and there is a no template complex and the template has an intense band approximately in the 800 bp region and that shows good quality sequence. The quality of DNA that can be checked through the measurement of the concentration in microgram per ml by (A260-A320)x the factor of dilutionx 50 microgram per ml (Ding, et al, 2018). This reading can conclude whether the 800 bp sequence is perfect for the PCR, this shows an intense dark band and by measuring the band intensity in different optical density the template purity can be checked. reaction or not and the primers that can be designed through different online tools that can provide a perfect primer for a template sequence.
Q2.a.
There are two different chemistries that exist for running a QPCR and those are the detection based on SYBR green and detection based on TaqMan.
Taq Man is more preferable chemist than the SYBR green because the latter has a tendency of producing primer dimer and also has the lack of specificity (Gupta et al, 2017). This also has the tendency to overestimate the concentration of target and that’s why for completing the QPCR Taq Man is preferred over SYBR green.
Chemistry of Taq Man:
The oligonucleotide with the fluorescent probe that binds to the 5’ end quench at the 3’ end. The 5’ end cut down due to the 5’ nuclease activity of the polymerase. That releases the fluorescent. The intensity of the released fluorescent depicts the rate of cleavage in the cycle. More the intensity of fluorescent, more the attachment to the presence of target sequence.
Chemistry of SYBR Green:
The intensity of the fluroscent increased with the accumulation of thye PCR product. This can form a primer dimer complex and also have the lack of overestimation of the target component.
Q PCR generates the double delta values that need the housekeeping and the gene of interest in both the control and the experimental (TE, HE) conditions and the average values of experimental and control (TC, HC) are calculated and then the delta values are calculated (TE-HE and TC-HC) and also the delta Ct values are calculated and to evaluate double delta Ct delta CTE - delta CTC performed.
As it is known that the qPCR is conducted for the quantification of the amount of target sequence and to make a comparison between the experimental and control cells (Zhou et al, 2017). The lower the Cq value the higher the higher the target sequence and higher Cq value indicates lower amount of nucleic acid.
The average control Ct values of the gene of interest is 12.15 and the reference cell is 12.125 and the Stimulated cells have 16.2 and 11.8 respectively for GOI and Ref Gene.(Prencipe, et al, 2020) the ref gene has higher target sequence content and as one factor of the control cells undetermined but the control cells contain (GOI) more target nucleic acids.
M.Sc. Molecular Biology and Biotechnology Coursework: Part II
Section 2:
Using the NCBI ORF founder and setting the ORF length parameter as mentioned in the query a ORF region is identified -
This has the positive strand and is situated in the frame 1, starts at 109 sequence and stops at 660 and the length is nt/aa, 552/183.
Table of restriction enzymes with the restriction sites in “pETSHU” MCS-
Restriction enzymes listed clockwise sequence
Site of restriction
End type
Product after cutting
EcoR V
5’ GATATC 3’
3’ CTATAG 5’
Blunt end producers
5’ GAT^ATC 3’
3’ CTA^TAG 5’
Produce blunt end by generating a cut within TA in both sequences.
EcoR I
5’GAATTC3’
3’CTTAAG5’
Sticky end generator
5’G^AATTC3’
3'CTTAA^G5' generates a cut on the prior site of the 5 terminal G in both sequences and produces a sticky end.
XhoI
5’ CTCGAG 3’
3’ GAGCTC 5’
Sticky end producer
5’ C^TCGAG 3’
3’ GAGCT^C 5’
Generate a cut before C in both sequences and that is towards the 3 prime ends.
EagI
5’ CGGCCG 3’
3’ GCCGGC 3’
Sticky end producer
5’ C^GGCCG 3’
3’ GCCGG^C 5’
Generate a cut before C in both sequences and that is towards the 3 prime ends.
SmaI
5’ CCCGGG 3’
3’ GGGCCC 5’
Blunt end producers
5’ CCC^GGG 3’
3’ GGG^CCC 5’
Generate a blunt end by making a cleave in between the sequences.
BamHI
5’ GGATCC 3’
3’ CCTAGG 5’
Sticky end producer
5’ G^GATCC 3’
3’ CCTAG^G 5’
Generate a cut in the prior position to G in both strands towards the 3 prime ends.
Table 1: Table of restriction enzymes with the restriction sites in “pETSHU” MCS
(Source: self made)
Those enzymes are restriction digestion enzymes that act on different restriction digestion sites and create different digestion fragments (Nell et al, 2020). There are two types of fragments found and those are Sticky end and blunt ends. Sticky end producers have the advantage over the blunt end producers and that is they do not need any help of adapters to ligate with any ligation site whereas, in blunt end products there is a additional step of adapters need to ligate the molecule of the gene of interest inside any vector’s ligation site of that particular restriction enzyme’s recognition site. Here in this vector there are two blunt end producers identified one is EcoRV and SmaI and the sticky end producers are EcoRI, XhoI, EgGI and BamHI.
Site of restriction:
The restriction site is found within the ORF, non overlapping site that codes 100 amino acids. These have the GC content of 465 and the AT content of 54%. There are a total 42 single cutters recognized with their cutting positions. 24 multiple site cutters are also recognized. The protein sequence in between BanII and EcoRV is-
The restriction enzyme cutting site is marked as a red cross on the ORF sequence depicted in figure 7. This sire has the recognition sequence for EagI that generates a sticky end after cutting (Szewczuk et al, 2021). Cutting also can be generated in the EcoRI site too.
There are two restriction enzymes chosen for the vector DNA and two chosen for the amplified cDNA. For the Q gene EagI and in EcoRI. Those two restriction enzymes are chosen that have their restriction recognition sites on the Qgene sequence. Those enzymes have the ability of producing sticky ends. A single cut or a double cut can be generated using these two restriction digestion enzymes.
These genes are also present within the MCS of the vector where two types of fragments are found and those are Sticky end and blunt ends. Sticky end producers have the advantage over the blunt end producers and that is they do not need any help of adapters to ligate with any ligation site whereas, in blunt end products there is a additional step of adapters need to ligate the molecule of the gene of interest inside any vector’s ligation site of that particular restriction enzyme’s recognition site. It is preferred to choose the same restriction enzymes to create cuts in both the vector and the gene of interest and that makes the ligation purpose more easy and helps to ligate the DNA fragment in a particular direction. The directional ligation is the strategy that helps to ligate a gene of interest in a particular position and orientation (Mendonça et al, 2019). This ligation also benefited from the inability to ligate with each other. While a single type of restriction enzyme digestion is used to make a fragment inside the sequence then there is a chance of being ligated with each other in the ligation reaction and that can generate a non transformed type of vector and the gene of interest remain as separated while when two different restriction enzyme sites are chosen then it has two different sequence of sticky end produced and that prevent the gene of interest from ligation (Moradpour et al, 2017). That process is also the same for the vector vector can also become ligate if a single RE is recognized then it can ligate again without taking the gene of interest. This is the benefit of choosing two different restriction enzymes for digestion and that also helps to proceed the work more correctly while this selection of two different enzymes eliminates the XhoI RE site from the vector MCS site.
M.Sc. Molecular Biology and Biotechnology Coursework Part III
Section Three
Expression of Q-gene into the vector pETSHU plasmid and expression under the promoter of T7:
Protein expression in the chosen expression system:
The expression system of T7 is in the recombinant DNA technology and this can be carried out through E.coli. This is used as a most popular system for the expression of recombinant types of proteins. The annotation and sequencing of the T7 genome of T7 bacteriophage the RNA polymerase of T7 is used along with the T7 promoter and that can transcribe copious amounts of any gene. Especially in the expression vector of pET a promoter of T7 and the gene of interest is placed downstream of the promoter (Pereira et al, 2018) . In any relevant host cell of E.coli the expression vector is transferred. In E.coli cells the cellular chromosome exists and that helps to express the T7 RNA Polymerase. This polymerase is mainly responsible for the beginning of the transcription with the T7 promoter that is present in the vector. Lac promoter controls the expression of the T7 gene (Moremen et al, 2018). The RNA polymerase can bind to the promoter and express the vector and transcribe the gene of interest. Lac promoter acts in the presence of lactose and to make more active expression IPTG (identical to lactose) used as an inducer that mimics allolactose structure. This is the way of expressing the downstream gene of interest and the expression of protein under the control of T7 promoter takes place. That is much faster , approximately 8x faster than the expression with the E.coli RNA polymerase.
Experimental scheme for collect the target protein from yield:
Protein purification is a series of different processes that can help to make a isolation results from a complex sample mix. Proteins are isolated from different tissues and cells of a whole organism. That is an important event for the specification of functions with different structures. That also highlights different events of the protein-protein interactions. This also separated out the protein and non-protein parts from a mixture and separates the protein of interest from different proteins. This separation is based on the size of the proteins, proteins physic-chemical properties and also as per their affinity towards other components. This process is termed as the isolation of proteins.
Protein purification and Q-protein isolation:
Stage: 1
Loading a protein’s simple Mixture
Methodology
Start from the beginning in the mentioned software. Complex Mixture was selected as the chosen mixture. Then within 1 to 60 proteins protein 10 was selected for the purification. The enzymatic activity of this protein 10 was that it can be stable for several hours up to 50 degree C and it has the pH within 3.5 to 11.5.It shows that the protein was 959 mg and the enzyme was 10200 and it has 100% yield. For the next experiment for gel filtration Sephadex G-50 used as the media. The gel filtration curve appeared.
Mechanism
1. Start from the beginning in the mentioned software
2. Complex Mixture was selected as the chosen mixture
3. Then within 1 to 60 proteins protein 10 was selected for the purification
4. The enzymatic activity of this protein 10 was that it can be stable for several hours up to 50 degree C and it has the pH within 3.5 to 11.5
5. It shows that the protein was 959 mg and the enzyme was 10200 and it has 100% yield
6. For next experiment for gel filtration Sephadex G-50 used as the media
7. The gel filtration curve was appeared
Rational
This method helps to do different experimental approaches with the protein that we need to be extracted and a 2D gel image helps to the changes of the fraction number with the increasing O.D.
Table 1: Stage 1
(Source: Self made)
Stage: 2
Agin that gel filtration in another gel matrix
Methodology
Start from the beginning in the mentioned software. Complex Mixture was selected as the chosen mixture. Then within 1 to 60 proteins protein 10 was selected for the purification. The enzymatic activity of this protein 10 was that it can be stable for several hours up to 50 degree C and it has the pH within 3.5 to 11.5.It shows that the protein was 959 mg and the enzyme was 10200 and it has 100% yield. For the next experiment for gel filtration Sephadex G-100 used as the media. The gel filtration curve appeared.
Mechanism
1. Start from the beginning in the mentioned software
2. Complex Mixture was selected as the chosen mixture
3.Then within 1 to 60 proteins protein 10 was selected for the purification
4.The enzymatic activity of this protein 10 was that it can be stable for several hours up to 50 degree C and it has the pH within 3.5 to 11.5
5.It shows that the protein was 959 mg and the enzyme was 10200 and it has 100% yield
6.For next experiment for gel filtration Sephadex G-100 used as the media
7.The gel filtration curve was appeared
Rational
The peaks are higher while Sephadex G-100 applied for the gel filtration. That spread in more fractions.
Table 1: Stage 2
(Source: Self made)
Stage: 3
Ion Exchange Chromatography
Methodology
Start from the beginning in the mentioned software. Complex Mixture was selected as the chosen mixture. Then within 1 to 60 proteins protein 10 was selected for the purification. The enzymatic activity of this protein 10 was that it can be stable for several hours up to 50 degree C and it has the pH within 3.5 to 11.5.It shows that the protein was 959 mg and the enzyme was 10200 and it has 100% yield. Then Selected the ion Exchange Chromatography.
Mechanism
1. In the separation procedure the Q-sepharose was selected
2. pH gradient used as the elution system
3. For Equilibration pH 7 was selected
4. Start with the gradient 6 and end with 7
Rational
This method is used for the purification of protein and that also shows that peaks at different OD and in different pH. At neutral pH the number of fractions increased exponentially and at pH 6 that steady up to 60 fractions. This protein has a wider range of pH and it is more stabilized at the higher pH.
Table 1: Stage 3
(Source: Self made)
Biological activity:
This gene encodes the AP2 factor of transcription that played an important role in “demonstration of polyploidy wheat” (Zhao et al, 2018). This gene also find in multiple organisms and the MSA result -
References:
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