Difference between revisions of "Part:BBa K4586015"
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==Characterization By Mutational Landscape== | ==Characterization By Mutational Landscape== | ||
| − | In order to optimize the function of our parts, we've used the concept of Directed Evolution through applying different mutations and measuring the effects of these mutations on their evolutionary epistatic fitness. As displayed in the chart below, the mutation (N80K) shows the highest epistatic fitness, while the lowest score was associated with the mutation (N99F). | + | In order to optimize the function of our parts, we've used the concept of Directed Evolution through applying different mutations and measuring the effects of these mutations on their evolutionary epistatic fitness. As displayed in the chart below, the mutation (N80K) shows the highest epistatic fitness, while the lowest score was associated with the mutation (N99F,P91C,C92I). |
<html><div align="center"style="border:solid #17252A; width:80%;float:center;"><img style=" max-width:850px; | <html><div align="center"style="border:solid #17252A; width:80%;float:center;"><img style=" max-width:850px; | ||
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lang=EN style='font-size:11.0pt;line-height:115%'>Figure . An illustration of the effects of different mutations on the Epistatic Fitness of L7ae. | lang=EN style='font-size:11.0pt;line-height:115%'>Figure . An illustration of the effects of different mutations on the Epistatic Fitness of L7ae. | ||
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| + | ==Experimental Characterization== | ||
| + | In order to amplify this DNA part, we used PCR amplification to reach the desired concentration to complete our experiments using specific forward and reverse primers, running the parts on gel electrophoresis as this part presents in lane (P4) including CD63 an L7Ae, and then measuring the specific concentration of the running part using Real-Time PCR as shown in the following figure. | ||
| + | <html><div align="center"style="border:solid #17252A; width:80%;float:center;"><img style=" max-width:850px; | ||
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| + | We performed the double digestion method for this part in the prefix and suffix with its specific restriction enzyme and applied this part to gel electrophoresis as shown in the following figure lane (P4). | ||
| + | <html><div align="center"style="border:solid #17252A; width:80%;float:center;"><img style=" max-width:850px; | ||
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| + | After the ligation step, we did a culture of the ligated product to specifically select the optimum colonies to screen it using Colony PCR to make sure that our parts were correctly ligated in the plasmid. This figure shows the Cell culture plate of transformed pCDNA vector 2 containing insert parts. | ||
| + | This plasmid contains Loading system(CD63-L7Ae)-exosomal receptor(LAMP2B-anti-CD19)-connexin(CX43) | ||
| + | <html><div align="center"style="border:solid #17252A; width:80%;float:center;"><img style=" max-width:850px; | ||
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| + | "src="https://static.igem.wiki/teams/4586/wiki/results/2.png"> | ||
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==References== | ==References== | ||
Russo, G. et al. (2005) Biochem J. 385, 289-299. | Russo, G. et al. (2005) Biochem J. 385, 289-299. | ||
Latest revision as of 13:59, 12 October 2023
L7Ae
Part Description
L7Ae is a 50S ribosomal protein that is considered an RNA-binding protein. It has the ability to recognize its K-turn motif or C/D box in ribosomal RNA and can be used to build more complicated genetic circuits that are regulated at both the translational and transcriptional levels as well as as an adapter for RNA binding in the extracellular vesicles.
Usage
Our team uses this part in loading our cargo into the exosomes. By binding to the C/D boxes to load a specific RNA into the vesicles, we fused it to RNA-motifs, which are bound specifically by RNA-binding proteins as shown in figure 1. and figure 2.
Figure 1: This figure illustrates the mechanism of loading our therapeutic agent in the form of mRNA selectively and efficiently into our engineered exosomes secreted form the MSCs,this loading is done through labeling the gene of interest with C\D box a hairpin structure IN THE 3` end this box have high affinity to the RNA binding protein L7Ae that is expressed on the internal surface of the engineered exosomes membrane conjugated to his tagged CD63 protein that is a natural highly expressed transmembrane protein within the exosomes.
Figure 2: This figure shows the design of the biological circuit expressing our loading system on the exosomes membrane ,this system consists of two main component ,First the RNA binding protein L7Ae conjugated to the second component, which is CD3 a transmembrane protein that is naturally expressed on the exosomes membrane.
Literature Characterization
The study made Western blot analysis and confocal immunofluorescent analyses of different cell lines using the L7a (E109) antibody.
By using Ribosomal Protein L7a (E109) Antibody, the study made Western blot analyses of extracts from various cell lines.
The study used the ribosomal protein L7a (E109) antibody, which appeared green, to make a confocal immunofluorescent analysis of COS cells. They labeled actin filaments with Alexa Fluor phalloidin 555, which appear red. DRAQ5 #4084 (fluorescent DNA dye) appeared as a blue pseudocolor. The antibody stains ribosomes at their biogenesis location in nucleoli.
Characterization By Mutational Landscape
In order to optimize the function of our parts, we've used the concept of Directed Evolution through applying different mutations and measuring the effects of these mutations on their evolutionary epistatic fitness. As displayed in the chart below, the mutation (N80K) shows the highest epistatic fitness, while the lowest score was associated with the mutation (N99F,P91C,C92I).
Figure . An illustration of the effects of different mutations on the Epistatic Fitness of L7ae.
Experimental Characterization
In order to amplify this DNA part, we used PCR amplification to reach the desired concentration to complete our experiments using specific forward and reverse primers, running the parts on gel electrophoresis as this part presents in lane (P4) including CD63 an L7Ae, and then measuring the specific concentration of the running part using Real-Time PCR as shown in the following figure.
We performed the double digestion method for this part in the prefix and suffix with its specific restriction enzyme and applied this part to gel electrophoresis as shown in the following figure lane (P4).
After the ligation step, we did a culture of the ligated product to specifically select the optimum colonies to screen it using Colony PCR to make sure that our parts were correctly ligated in the plasmid. This figure shows the Cell culture plate of transformed pCDNA vector 2 containing insert parts. This plasmid contains Loading system(CD63-L7Ae)-exosomal receptor(LAMP2B-anti-CD19)-connexin(CX43)
References
Russo, G. et al. (2005) Biochem J. 385, 289-299. Sequence and Features
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