All biobricks were synthesized by Integrated DNA Technologies.
The first five biobricks are tRNA genes from the genome of Vibrio natriegens. The five rarest tRNA genes were identified and analyzed with different software as described in detail on the part pages. With the help of these tRNAs, the translation speed should be increased, resulting in increased protein expression. The tRNA genes contain 50 nucleotides of the upstream sequence to minimize the influence on later tRNA maturation. Downstream only a few nucleotides were included depending on the position of unwanted terminator loops.
To express the tRNA genes, they were cloned into a self-designed plasmid backbone (ptRNA_backbone). The plasmid uses a p15A origin of replication, making this plasmid compatible with the popular ColE1/pMB1/pBR322/puc origin of replication and contains a tetracycline resistance Furthermore, regulatory elements for tRNA transcription such as the rrnA P1 promoter (from Vibrio natriegens1) and the rrnA terminator (from E. coli K12) were added. With this operon, the transcription of the tRNAs should be proportional to the growth rate and adjust to the cellular levels of the remain protein synthesis machinery2.
DNA-RNA-Hybrid-Adapter for tRNA quantification
For tRNA level measurements, a DNA/RNA hybrid adapter was designed that binds specifically to the 5' terminus of the mature tRNA. Using a primer that binds to the adapter, the desired tRNA can be amplified in a PCR reaction. Individual tRNA levels can be quantified with the use of a DNA intercalating dye.
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Additionally, the Biobricks GFP (http://parts.igem.org/Part:BBa_E0040) and sfGFP (http://parts.igem.org/Part:BBa_I746916) have been improved by optimizing them for Vibrio natriegens. The sequences were analyzed for rare codons and then exchanged with more frequently used ones. This means that no classical codon optimization was performed, but rare codons, which could have a strong influence on the expression strength, were exchanged. By doing so, we wanted to increase the expression of GFP and sfGFP in Vibrio natriegens and thus provide a better measurement tool. The disadvantage of codon optimization is that the bottleneck of the availability of rare tRNAs is not fixed but only bypassed. Nevertheless, it should be shown that codon optimization has a similar effect as increasing the availability of rare tRNAs.
|1||BBa_K3014000||Coding||Vibrio natriegens tRNA AGA||Benedikt Schober, Jan Müller, Kai Schülke||135|
|2||BBa_K3014001||Coding||Vibrio natriegens tRNA AGG||Benedikt Schober, Jan Müller, Kai Schülke||135|
|3||BBa_K3014002||Coding||Vibrio natriegens tRNA CGG||Benedikt Schober, Jan Müller, Kai Schülke||160|
|4||BBa_K3014003||Coding||Vibrio natriegens tRNA TGC||Benedikt Schober, Jan Müller, Kai Schülke||161|
|5||BBa_K3014004||Coding||Vibrio natriegens tRNA TCC||Benedikt Schober, Jan Müller, Kai Schülke||151|
|6||BBa_K3014006||Plasmid_Backbone||ptRNA_backbone||Benedikt Schober, Jan Müller, Kai Schülke||2161|
|7||BBa_K3014007||Coding||Codon optimized GFP for Vibrio natriegens||Benedikt Schober|
|8||BBa_K3014008||Coding||Codon optimized sfGFP for Vibrio natriegens||Benedikt Schober||720|
|9||BBa_K3014009||Measurement||DNA-RNA-Hybrid-Adapter for tRNA quantification||Katharina Hofer, Sini Münßinger, Jan Seeger, Gabriele Kepp||130|
|10||BBa_K3014010||Terminator||Terminator of rrnA operon V. natriegens||Jan Müller, Kai Schülke||54|
|11||K3014011||Regulatory||V. natriegens rrnA P1 Promoter||Benedikt Schober, Jan Müller, Kai Schülke||71|
|12||BBa_K3014012||Coding||Vibrio natriegens tRNA construct||Benedikt Schober, Jan Müller, Kai Schülke||915|