Part:BBa_K1791002:Experience

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Applications of BBa_K1791002

GENERATION OF RNA USING RIBOZYME AFFINITY PURIFICATION: Comparing high and low MS2 Expression BBa_1791001 (high) Vs. BBa_1791002 (low)

We used this part to purify RNA utilizing our Ribozyme Affinity Purification strategy. Two separate constructs were employed; one construct expresses amino-terminally 6xHistidine-tagged MS2 coat-protein under the control of a T7 promoter and High ribosome binding site (RBS) (termed High RBS BBa_1791001), while the second construct expresses the tagged MS2 under control of a T7 promoter and weak RBS (Low RBS BBa_1791002). In this way, we hoped to determine the optimal amount of MS2 expression required for effective purification of our desired RNA species by modulating the translation efficiency of MS2 coat protein. Both of the constructs also contain a theophylline aptazyme coding module that is also under control of a T7 promoter.

Initially, our constructs were cloned into the pSB1C3 backbone vector and then transformed into BL21(DE3) Escherichia coli cells. Induction of protein and target RNA expression was initiated simultaneously by addition of 1 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) that results in overexpression of T7 RNA polymerase.

Media:Figure 1. Low RBS RAP SDS-PAGE.jpg

Media:Figure 2. High RBS RAP SDS-PAGE.jpg

As expected, induction of the High RBS construct resulted in strong expression of MS2 coat-protein, whilst the Low RBS construct showed markedly less expression with MS2 not being easily distinguishable against the E. coli protein background, in the latter (Fig. 1). Nonetheless, upon performing Ni2+-affinity chromatography followed by SDS-PAGE analysis of samples, both High RBS and Low RBS showed detectable amounts of purified MS2 protein (Fig 2). Interestingly however, the yield of purified MS2 protein from the Low RBS construct was similar to that of the High RBS construct. Indeed, we observed a considerable amount of the MS2 protein in the insoluble fraction of the High RBS construct purification and we predict that this loss resulted in the reduced yields of MS2. In the future, other expression conditions and/or MS2 isoforms will be tested to improve the yield of soluble protein.

Next, we wanted to determine if we had successfully co-purified the aptazyme transcript during purification of MS2 protein. Consequently, we performed phenolic extractions on one tenth of each protein purification sample and then examined them for the presence of RNA on denaturing urea polyacrylamide gels. Interestingly, while the Low RBS samples did not show detectable aptazyme transcripts (230 to 250 nucleotides, nt), elusions from the High RBS purification showed a clear enrichment of RNAs with expected length for the aptazyme transcript (Fig. 6). Most strikingly, the predicted ratio of the full-length to cleaved aptazyme appears to be improved over our in vitro transcribed aptazymes (~70% full-length versus ~30% cleaved). We take these results to suggest that cellular conditions during expression in vivo and/or differences in RNA polymerase incorporation rate affect the folding of the aptazyme and consequently the frequency of cleavage. It is also interesting to note that we detected a small amount of the small RNA cleavage product after RNA purification. Because the small RNA fragment does not contain the MS2 binding region, we take this to suggest that cleavage events giving rise to the fragments occurred sometime during or after purification. If this is true, then the actual amount of full-length transcript from the in vivo expression may be even higher than the predicted ~70%.

Media:Figure 3. Low RBS RAP Urea PAGE.png

Media:Figure 4. High RBS RAP Urea PAGE.jpg

Finally, we wanted to determine whether the full-length in vivo purified aptazyme transcripts could be induced to cleave via addition of theophylline. Purified RNAs were incubated with theophylline concentrations from 1μM to 1mM and analyzed by denaturing PAGE. In contrast to the previous cleavage assays using in vitro transcribed aptazyme, we did not detect any significant cleavage of the in vivo purified transcript (Fig. 7). Because conditions for aptazyme cleavage may require different buffer conditions (such as pH, salt and magnesium), in the future we will attempt to induce cleavage using a variety of reaction conditions.

Media:Figure 5. theophylline concentrations from 1μM to 1mM.jpg

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