The Central Dogma Of Molecular Biology Accounts Biology Essay

  Student Number: 210310399

TA: Dave

STudying the expression of a pure and function t7 RNA polymerase in an invitro translation of mutant pokeweed antiviral protein

Introduction

The central dogma of molecular biology accounts for the detailed residue-by-residue transfer of sequential information through different stages at the genomic levels. It states that information cannot be coded back from protein to either DNA or RNA; however, RNA can be reverse transcribed into DNA (Hurwitz 2005). Transcription is the process through which RNA is made from DNA, while protein is synthesized through translation of the transcribed RNA (Hurwitz 2005). This partially reversible sequential order of synthesis (DNA - RNA - Protein) makes up the fundamentals of molecular biology. The synthesis of protein along with its cellular interactions is vital to various regulatory signalling pathways such as the Wnt and AKT pathways (Martin and Esposito 2005).

The ever-growing field of Biotechnology implements various techniques to study a vast array of interactions in biology. Using autoradiography, this experimental study investigates the expression of a pure and functional T7 RNA polymerase in order to transcribe and translate a mutant strain of the Pokeweed Antiviral Protein (PAPx). PAP is a 29 KDa ribosome-inhibiting protein (RIP) which acts by depurinating ribosomes at the alpha-sarcin/ricin loop of the large rRNA, ultimately resulting in the inhibition of translation (Tumer and Hwang 1997). pBluescript plasmid, KH014, containing an active site mutant of the pokeweed antiviral protein is transformed into E.coli (DH5α) bacterial cells. However, the site mutation will, upon transcription of T7 polymerase, allow for the subsequent production of PAPx.

Plasmids are important tools when expressing specific genes and are used in genetic and biotechnology labs. The gene is inserted into copies of a plasmid that makes cells resistant to a particular antibiotic (Byrne and Mason 2009). In addition, a multiple cloning site region consisting of common restriction sites like HindIII and EcoRI, is present to allow for easy integration of DNA fragments (Tumer and Hwang 1997). Thereafter, the cells are transformed into bacteria and exposed to the antibiotic. Only those cells that have taken up the plasmid survive this ampicilin selection as the plasmid renders them resistant. These bacterial cells can then be grown, harvested, lysed in large quantities (Byrne and Mason 2009).

Autoradiography creates an image on x-ray film by utilizing the properties of particles that are radioactive . Particularly, this technique relies on emissions of alpha and beta particles and gamma rays released by the decay of radioactive substances within the sample, rather than an outside source (Peterson and Bray 1968). The resulting image will allow for determination of the relative concentration of radioactive material and its distribution. This laboratory study implemented the use of 35S, a radioactive probe to allow for the detection of the protein of interest (Johnston et al. 1990). This was incorporated at the amino acid methionine. This technique can be applied towards the detection of DNA, RNA and protein and serves as a cellular marker in some cases (Jin & Lloyd 1997).

T7 RNA polymerase is a 99 kDa (approx.) polymerase from the T7 bacteriophage that plays a key role in the catalysis of RNA synthesis in the 5'→ 3' direction and is also dependent on the presence of DNA. It is promoter specific as it only transcribes DNA downstream of the T7 promoter which nowadays, is already incorporated into the plasmid. This study uses T7 RNA polymerase to conduct an in vitro transcription of DNA to RNA, upon which, the transcribed RNA is used as the template strand to conduct an in vitro translation experiment, leading to the synthesis of PAPx. PAPx is radioactively labelled with a 35S probe, specifically at methionine.

This study begun with BL21 E. coli cells being transformed with a plasmid that contained cDNA that encodes for T7 RNA polymerase. Competent BL21 cells are preferentially used due to their heightened expression of protein by basically sending the expression machinery into "over-drive" (Kleber-Janke et al. 2000). Another compulsory component of this machinery is the presence of a magnesium (Mg2+) ion as cofactor. The expression of T7 RNA polymerase is facilitated and controlled by The lac promoter and operator. LacI controls the lac operon, which ultimately expresses a translated tetrameric protein that binds to the operator portion on DNA. This inhibits RNA polymerase from binding and therefore it is repressed. The affinity for the operator region is reduced when an inducer binds to regions on the tetramer proteins. This results in its removal from the DNA strand. Ultimately, this permits binding to the sequence of interest by the endogenous bacterial e.coli cells which subsequently transcribe genes. The induction of expression was done through the addition of IPTG (Johnsrud 1978).

Within the plasmid, there was a his-tag added that ultimately was co-transformed into the BL21 bacterial e.coli cells. This tag was essentially co-expressed and bound to the T7 polymerase when it went through the process of transcription and subsequently translation. The affinity purification was conducted using Ni-NTA resin. This resin binds to the His-tag which is bound to the T7 polymerase and they were pulled down through a process called gravity-flow chromatography. The technique functions on the affinity between Histidine and Nickel ions from the resin (Kleber-Janke et al. 2000). Lysate samples were also added to imidazole elution solution. Imidazole is commonly implemented into the His-tag purification protocol as it plays a competing role since it comprises of the histidine side chain. In addition, it works by inhibiting the bond between his-tag and Nickel as a means of eluting purified protein (Kleber-Janke et al. 2000). These products, in their purified state, can be ultra-filtered using a Centricon 50 filter. The purification of polymerase is followed by the isolation of the KH014 plasmid DNA that had been transformed into DH5α E. coli cells. This plasmid contains the PAP gene in the mutated form. This plasmid is used as the template DNA for the T7 polymerase to transcribe in the in vitro transcription step. RNase A was also added to the solution to cleave single-stranded RNA to degrade RNA but not the plasmid as well. Using NOAc and a 100% ethanol, the RNA was precipitated from the solution. (Miller et al. 1999). HindIII, a restriction enzyme, was used on the isolated plasmid DNA, to digest it through the night at 37 degrees Celsius. The cut occurs at 5'---A/AGCTT---3' in order to linearize the circular DNA in order for it be considered a substrate for T7 RNA polymerase to transcribe so that T7 RNA polymerase can use it as the substrate. Phenol:Chloroform:Isoamyl alcohol was used after the overnight digestion to remove the restriction enzyme, HindIII and other interfering proteins that may be present (Miller et al. 1999).

Materials and Methods

The protocol was implemented as per the Biotechnology experimental laboratory handbook provided by the Department of Biology at York University [1] . Changes to the protocol include page 25, wherein step 2 included 400 uM IPTG; page 26, steps 5, 6 and 7 were done at 3000 rpm and steps 6 and 7 included 3 minutes of centrifugation; page 26, nickel resin was washed with 1mL of lysis buffer prior to use; page 27, steps 7 and 9 included using 10 uL of 2X SDS; page 27, in step 10, 500 uL of DEPC H2O was added to the filter and step 11 was done till 100 uL remained in the filter; page 28, step 2 included adding 10 mL of 70% ethanol while in step 5, a 1:499 dilution was made; page 28, step 1 and 2 included adding 500 ul of wash solution and centrifugation took place till 100 uL remained in filter; page 30, step 6 included dilution of 1:499; page 32, step 4 included dilution of 1:499; and page 34, step 5 included adding 15 uL of 2X SDS.

Discussion

This experimental study sought out to investigate the efficiency of a pure and functional T7 RNA polymerase in the transcription and subsequent in vitro translation of BL21 E. coli cells transformed with KH014 plasmid, which contains an active site mutation in PAP (PAPx). This modification ultimately allows for the progression of translation, which was done by incorporating a radioactive probe (35S), in order to accurately visualize nascent protein post-translation. The hypothesis is that provided all steps of the transcription and translation process are conducted in an efficient manner, the T7 RNA polymerase would have successfully proven its functionality by transcribing the isolated linear cut DNA. The ribosomes and protein producing factors provided by the reticulocyte lysate would have then translated the mRNA transcript into PAPx. As it is shown through Figures 1.0 to 4.0, although the polymerase exhibited functional attributes, as shown through its proper transcription of the DNA yielding a mRNA transcript, the subsequent translation step failed, yielding no PAPx. Until the translation step, all molecular weights of template DNA coding for PAPx, T7 RNA polymerase and BSA were comparable to their true values (3.7 kilobases, 99 kDa and 66 kDa respectively).

As a precursor to the transcription of the isolated linear cut DNA of interest, the T7 RNA polymerase was purified. After being grown in bacterial BL21 cells and induced with IPTG, the cells were harvested and given to the students. These were then lysed, initiating the affinity purification process. As shown in figure 1.0, T7 RNA polymerase was affinity purified using a His-tag. This allowed for purification through His' affinity to Nickel (Ni2+). It is seen that the p7702 Broad Range Protein marker did not resolve well on the 10% SDS-PAGE polyacrylamide gel and that poses some discrepancies when qualitatively trying to quantify the concentration of T7 polymerase. Furthermore, the cell lysate sample was taken post-sonication to confirm that the cells have been lysed. In addition, when cells are incubating too long in lysis buffer or sonication has taken place at too high of an amplitude or for an extended period of time, there are negative functional consequences downstream of the purification. Also, the crude sample was taken out after centrifuging the sample at high speeds. This is because the protein that is sought remains in the soluble layer of the solution. As seen in lane 4 compared to lane 3, there is less aggregation as we have isolated the soluble layer which contains the proteins and other sub cellular fractions. After incubating the lysate with nickel beads for an hour, wash buffer was added and protein bound to the nickel beads by virtue of the His-tag were isolated.

Different concentrations of imidazole (1mM and 10 mM) was used to wash the sample. It is observed that contaminants existed in both samples, although the concentration decreased in the second wash as the imidazole concentration increased, which lead to a greater purification stringency. Using 250 mM concentration of imidazole, the sample was eluted. When adding 250 mM of imidazole, it competes with the affinity between His-tagged protein and Nickel beads, ultimately breaking that bond and pulling down the His-tagged protein in the eluate. The protein was isolated and purified as shown in lane 7 compared to lanes 2 and 3. However, it is important to take note that contaminants still reside in the "purified" T7 polymerase. This can be accounted for by only conducting 2 washes and not confirming the lack of contaminants through a Bradford protein assay, wherein 5ul of sample is added to 100ul of Bradford reagent and washes are continually done till the 100ul sample does not turn blue anymore. I

n addition, lanes 8 through 10 consisted of dilutions (1:20, 1:10, 1:2) of the concentrated T7 RNA polymerase in lane 11. An important observation is that as the eluate is concentrated, the contaminants remain above the filter as they are large enough to not pass through. Furthermore, the importance of these dilutions lies in the purpose of lanes 2 and 3, wherein 0.5 ug and 1.0 ug of Bovine Serum Albumin (BSA) was added. BSA is often added to an SDS-PAGE gel as a means of qualitatively quantifying the concentration of the protein of interest. In this study, as a way to increase accuracy, dilutions were created to ensure that multiple samples could be compared to the BSA standards. It is apparent that even though the gel did not resolve well, the concentration of T7 RNA polymerase is 1.5 times the 1.0 ug BSA sample in lane 3 and therefore that is the visualized concentration of the polymerase. For the in vitro transcription, the final concentration of T7 RNA polymerase needs to be 1 mg/ml. The final purified sample still had contaminants present which has a chance of hindering other interactions from taking place; however, a plausible solution would be to run the eluate through an FPLC machine to attain a purified sample that has undergone the most stringent purification. In addition, it would be ideal to conduct an expression test (samples pre and post-IPTG induction) to evaluate the efficiency with which IPTG is being taken in by the bacterial cells.

After the purification of the T7 RNA polymerase, it is vital that the KH014 plasmid is isolated. The KH014 or pBluescript plasmid contains the gene that encodes for the mutant PAP along with a T7 promoter, which the polymerase will use in the transcription process. Prior to its use as a substrate for in vitro transcription, the circular plasmid DNA must be linearized through a single restriction enzyme digest. This was done by incubating the KH014 plasmid with HindIII restriction enzyme overnight at 37 degrees Celsius for optimal cutting at 5'---A AGCTT---3'. In the isolation steps, the sample was flushed with phenol:chloroform:isoamyl, after which a high speed centrifugation yields a phase separation. The cut linear DNA is isolated to the top aqueous layer and thus, that layer is taken out and 3M NaOAc and 100% ethanol were added prior to vortex and storage. This sample was then taken along with a sample of the positive control (circular plasmid DNA) and both were run on a 1% agarose gel along with a NEB 1 kB ladder. Figure 2.0 reveals an accurate representation of the postulated results for this step of the study. At 2.3 kilobases, the circular DNA, since it is supercoiled, runs at a faster rate than linear cut DNA. This is exhibited by Figure 2.0. Furthermore, unlike the linear cut DNA, the circular DNA exhibited a light smear downstream of the 2.3 kilobase band, which could be attributed to possible degradation; however, since the linear DNA is clean, it is a good sample to work with for the in vitro transcription.

Upon preparing the T7 polymerase and linear cut DNA, the in vitro transcription can be set up. The reaction set up increases linear DNA to act as the DNA template, which is vital as the T7 polymerase is a DNA-dependent polymerase. In addition, RNase inhibitor is added to ensure that nucleases do not attack the mRNA transcript after it has been transcribed. Furthermore, rNTPS are added to facilitate the reaction. Figure 3.0.A reveals the RNA transcript along with a 100 bp ladder run on a 7M urea/6% polyacrylamide gel and viewed under UV 254 nm light. As mRNA was only attained by one group, subsequent translation experiments were done with the mRNA presented in Figure 3.0.B. The most logical reason for mRNA transcript not being present is that despite adding RNase inhibitors, it is possible that an increased contamination would have negated any anti-inhibitory effects by the inhibitor. This would have subsequently degraded the mRNA that was transcribed. As the T7 RNA polymerase was purified well enough and the linear DNA was cut efficiently, the problem occurred specifically during the in vitro transcription. Nevertheless, for the purpose of continuing this investigation, group 7's mRNA transcript was used. This mRNA transcript is postulated to exhibit a band at 800 kb; however, figure 3.0.B reveals a band at around 900 kb. The reasons for an increased molecular weight are plentiful; however, perhaps incomplete processing yielded a greater molecular weight. The best way to avoid future contaminations is to conduct such an experiment in an RNase-free fume hood or isolated area which has been sterilized with chloroform.

Using the in vitro transcribed mRNA transcript from group 7, autoradiography is used to visualize an in vitro translated 35S-labelled protein. In the reaction mix, 17 ul of reticulocyte lysate was added to provide the protein factors and ribosomes required for translation. Since we isolated the mRNA transcript and took out the enzymes and T7 RNA polymerase, this mRNA transcript is added to a new mix in which 35S methionine, a radioactive probe is added. These samples were then run on a 12% SDS-PAGE polyacrylamide gel with the negative control including the sample minus any RNA transcript. In addition, an unknown positive control was added to ensure that the probe was being taken up by a known protein. As figure 4.0 reveals, only the positive control successfully worked. It is hard to dispute against the negative control as nothing should show in this lane, and nothing has done so; however, lane 3 consisted of what is postulated to be the radio-labelled protein. It is apparent that nothing showed up and that either the translation of the mRNA or the uptake of the radioactive probe failed; however, the positive control can attest to the reliability of the probe. In addition, the presence of the ribosomes and protein factors from the reticulocyte lysate could not have played the role they were meant to do by supplying the necessary factors for proper translation. In addition, the smears from Figure 3.0.B could also attest to the possibility of degraded RNA. After isolated the transcript, there is a chance that contamination could have introduced RNases. In addition, continual freezing and thawing also affects stability of transcript and protein ultimately affecting the autoradiogram and the visualization of the radioactively labelled protein. Furthermore, if the increased molecular weight in Figure 3.0.B could be related to the incomplete processing of the DNA when transcribing, there is a chance that an intron very early on in the sequence, that would not affect transcription, halts translation. In addition, the uptake of the radioactive probe will be compromised.

This experimental study sought out to transcribe the PAPx gene and subsequently translate the mRNA transcript into the PAPx protein; however, the experiment's design was poor as the purpose of the site mutant is abolished as this is an in vitro experiment. The effects of PAP and PAPx cannot be thoroughly investigated. Tumer and Hwang 1997 conducted a study where in they focused on specific site mutations and looked at the levels of PAP and PAPx. Their experimental study was successful as PAPx protein was successfully synthesized. There are many flaws including that of countless contaminants and insufficient purification steps that compromise the integrity of the results. Though it is very much possible to synthesize PAPx protein and view it after incorporating a radioactive probe, using this experimental design, it will not be a success. The design raises more questions than answers as an accurate protocol needs to be sought out.