Team:Jilin China/Basic Part
BASIC PART
Basic Part
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Abstract
This year, Jilin_China added 91 basic parts to the registry, including heat-inducible RNA thermosensors, heat-repressible RNA thermosensors, cold-inducible RNA thermosensors, cold-repressible RNA thermosensors and two different sfGFP. We have characterized and measured all of these parts, calculated their melting temperatures by using mathematical modeling, and successfully built a synRT toolkit that allows users to select the appropriate RNA thermosensors in artificial biological systems.
Our team's favorite basic part is the heat-inducible RNA-based thermosensor (BBa_K2541029) and will be introduced in detail below:
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RNA-based thermosensors
Heat-inducible RNA thermosensors
Heat-inducible RNA-based thermosensors are RNA-based genetic control systems that sense temperature changes. At low temperatures, the mRNA adopts a stem-loop that sequesters the ribosome binding site [Shine–Dalgarno (SD) sequence] within the 5′-untranslated region (5′-UTR). In this way, we could prevent ribosome binding and translation. At elevated temperature, the stem-loop melts, thereby making the ribosomes accessible to the ribosome binding site to initiate translation. There are some naturally-occurring RNA thermosensors, however, they have complicated secondary structures with multiple stems, loops and bulges, which makes them difficult to implement in engineered systems. Our team designed synthetic heat-inducible RNA-based thermosensors that are considerably simpler than naturally occurring thermosensors and can be exploited as convenient on/off switches of gene expression. Since they performed very well, we chose BBa_K2541029 as our favorite basic part, which was the most extraordinary one.
Heat-repressible RNA thermosensors
Most naturally-occurring RNA thermosensors are heat-inducible, which have long sequences and function by sequestering the ribosome binding site in a stem-loop structure at lower temperatures. Here, we also designed short heat-repressible RNA-based thermosensors. These thermosensors contain a single-strand RNA cleavage site for RNase E, an enzyme native to Escherichia coli and many other organisms, in the 5' untranslated region of the target gene. At low temperatures, the cleavage site is sequestered in a stem-loop and gene expression is unobstructed. Whereas at elevated temperatures, the stem-loop unfolds, and the mRNA is degradated and gene expression is turned off. These short, modular heat-repressible RNA-based thermosensors can be applied to the construction of complex genetic circuits, facilitating rational reprogramming of cellular processes for synthetic biology applications.
Cold-inducible RNA thermosensors
There are multiple families of cold-inducible proteins in prokaryotes, in which the most widely studied one are the CspA, which has been quite extensively investigated. There is a temperature-sensing region in the 5'UTR of CspA mRNA, which can regulate the accessibility of the translation initiation region by altering the advanced structure of mRNA. At low temperatures (<20℃), the 5’UTR of CspA mRNA can form an advanced structure called pseudoknot. Then the pseudoknot conformation exposes the Shine–Dalgarno (SD) sequence and it is beneficial to recruit ribosomes and somewhat less susceptible to degradation. At normal temperatures, due to thermodynamic instability, pseudoknot unfolds. As a result of that, 5’UTR of CspA forms a secondary structure, which sequesters Shine–Dalgarno (SD) sequence and further block translation initiation region and impedes translation. In our project, we have deleted the conserved region called the cold box located upstream of the 5'UTR of CspA mRNA, so that the expression of CspA is not regulated by its own negative feedback. The pseudoknot in the CspA mRNA contains four sets of base pairings, and its stability is temperature-regulated. We increased the number of base pairing or increased the GC content of CspA gene, which may increase the temperature threshold for pseudoknot unfolding. In addition, we also reduced the number of base pairing or reduced the GC content, which could make the temperature threshold for pseudoknot unfolding drop down. Hence, our team has successfully designed synthetic cold-inducible RNA thermosensors that are considerably simpler than naturally occurring CspA thermosensors and those cold-inducible thermosensors can be exploited as convenient on/off switches of gene expression.
Cold-repressible RNA thermosensors
These thermosensors contain a double-stranded (ds) RNA cleavage site for RNase III, an ribonuclease native to Escherichia coli, which could cleave dsRNA in a highly site-specific manner. This cleavage site was inserted in the 5' untranslated region of the target gene. At low temperatures, the mRNA stem-loop is stable and the RNase III cleavage site is exposed, leading to the degradation of the mRNA. At elevated temperature, the stem-loop will unfold and gene translation will occur unhinder. These short, modular cold-repressible RNA thermosensors can be applied to the construction of complex genetic circuits, facilitating rational reprogramming of cellular processes for synthetic biology applications.
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sfGFP_optimism (BBa_K2541400)
sfGFP (superfolder GFP), whose emission and excitation wavelength are similar to GFP, contains a higher fluorescence intensity and folding speed than GFP. Thus, we applied sfGFP as the reporter protein in our measurement device . However, the existing sfGFP (BBa_I746916) in the registry contains a BbsI endonuclease cleavage site, which is needed in Golden Gate assembly system in our program. In view of that, we designed a site-directed mutation of sfGFP (BBa_K2541401) by creating a double-base mutation to the BbsI recognition site without changing the amino acid sequence. sfGFP BBa_K2541401 won’t be digested during the assembly, so we also called it sfGFP for Golden Gate assembly.
We also performed a codon optimization sfGFP for prokaryote, and named it sfGFP_optimism. Then we designed a composite part, which contains Anderson Promoter J23104, RBS B0034 and sfGFP_optimism. We did experiments to compare it with sfGFP and sfGFP for Golden Gate assembly. Our results showed that the sfGFP_optimism has a higher fluorescence intensity than others, so we finally chose sfGFP_optimism as our reporter protein.
You can see the experiment results in the improvement page. Click Here! Since sfGFP has more advantages than GFP, and Goldengate assembly will be used by more researchers as an efficient and scarless assembly method in the future, so we decided to add sfGFP_optimism to the parts registry.
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Basic parts content
Part Name Part Number Heat-inducible RNA-based thermosensor-1 BBa_K2541001 Heat-inducible RNA-based thermosensor-2 BBa_K2541002 Heat-inducible RNA-based thermosensor-3 BBa_K2541003 Heat-inducible RNA-based thermosensor-4 BBa_K2541004 Heat-inducible RNA-based thermosensor-5 BBa_K2541005 Heat-inducible RNA-based thermosensor-6 BBa_K2541006 Heat-inducible RNA-based thermosensor-7 BBa_K2541007 Heat-inducible RNA-based thermosensor-8 BBa_K2541008 Heat-inducible RNA-based thermosensor-9 BBa_K2541009 Heat-inducible RNA-based thermosensor-10 BBa_K2541010 Heat-inducible RNA-based thermosensor-11 BBa_K2541011 Heat-inducible RNA-based thermosensor-12 BBa_K2541012 Heat-inducible RNA-based thermosensor-13 BBa_K2541013 Heat-inducible RNA-based thermosensor-14 BBa_K2541014 Heat-inducible RNA-based thermosensor-15 BBa_K2541015 Heat-inducible RNA-based thermosensor-16 BBa_K2541016 Heat-inducible RNA-based thermosensor-17 BBa_K2541017 Heat-inducible RNA-based thermosensor-18 BBa_K2541018 Heat-inducible RNA-based thermosensor-19 BBa_K2541019 Heat-inducible RNA-based thermosensor-20 BBa_K2541020 Heat-inducible RNA-based thermosensor-21 BBa_K2541021 Heat-inducible RNA-based thermosensor-25 BBa_K2541025 Heat-inducible RNA-based thermosensor-26 BBa_K2541026 Heat-inducible RNA-based thermosensor-27 BBa_K2541027 Heat-inducible RNA-based thermosensor-28 BBa_K2541028 Heat-inducible RNA-based thermosensor-29 BBa_K2541029 Heat-inducible RNA-based thermosensor-30 BBa_K2541030 Heat-inducible RNA-based thermosensor-31 BBa_K2541031 Heat-inducible RNA-based thermosensor-32 BBa_K2541032 Heat-inducible RNA-based thermosensor-33 BBa_K2541033 Heat-inducible RNA-based thermosensor-34 BBa_K2541034 Heat-inducible RNA-based thermosensor-35 BBa_K2541035 Heat-inducible RNA-based thermosensor-36 BBa_K2541036 Heat-inducible RNA-based thermosensor-37 BBa_K2541037 Heat-inducible RNA-based thermosensor-38 BBa_K2541038 Heat-inducible RNA-based thermosensor-39 BBa_K2541039 Heat-inducible RNA-based thermosensor-40 BBa_K2541040 Heat-inducible RNA-based thermosensor-41 BBa_K2541041 Heat-inducible RNA-based thermosensor-42 BBa_K2541042 Heat-inducible RNA-based thermosensor-43 BBa_K2541043 Heat-inducible RNA-based thermosensor-44 BBa_K2541044 Heat-inducible RNA-based thermosensor-45 BBa_K2541045 Heat-inducible RNA-based thermosensor-46 BBa_K2541046 Heat-inducible RNA-based thermosensor-47 BBa_K2541047 Heat-inducible RNA-based thermosensor-48 BBa_K2541048 Heat-inducible RNA-based thermosensor-49 BBa_K2541049 Heat-inducible RNA-based thermosensor-50 BBa_K2541050 Heat-inducible RNA-based thermosensor-51 BBa_K2541051 Heat-repressible RNA-based thermosensor-1 BBa_K2541101 Heat-repressible RNA-based thermosensor-2 BBa_K2541102 Heat-repressible RNA-based thermosensor-3 BBa_K2541103 Heat-repressible RNA-based thermosensor-4 BBa_K2541104 Heat-repressible RNA-based thermosensor-5 BBa_K2541105 Heat-repressible RNA-based thermosensor-6 BBa_K2541106 Heat-repressible RNA-based thermosensor-7 BBa_K2541107 Heat-repressible RNA-based thermosensor-8 BBa_K2541108 Heat-repressible RNA-based thermosensor-9 BBa_K2541109 Heat-repressible RNA-based thermosensor-10 BBa_K2541110 Heat-repressible RNA-based thermosensor-11 BBa_K2541111 Heat-repressible RNA-based thermosensor-12 BBa_K2541112 Heat-repressible RNA-based thermosensor-13 BBa_K2541113 Heat-repressible RNA-based thermosensor-14 BBa_K2541114 Heat-repressible RNA-based thermosensor-15 BBa_K2541115 Heat-repressible RNA-based thermosensor-16 BBa_K2541116 Heat-repressible RNA-based thermosensor-17 BBa_K2541117 Heat-repressible RNA-based thermosensor-18 BBa_K2541118 Heat-repressible RNA-based thermosensor-19 BBa_K2541119 Heat-repressible RNA-based thermosensor-20 BBa_K2541120 Heat-repressible RNA-based thermosensor-21 BBa_K2541121 Heat-repressible RNA-based thermosensor-22 BBa_K2541122 Heat-repressible RNA-based thermosensor-23 BBa_K2541123 Cold-inducible RNA-based thermosensor-1 BBa_K2541301 Cold-inducible RNA-based thermosensor-2 BBa_K2541302 Cold-inducible RNA-based thermosensor-3 BBa_K2541303 Cold-inducible RNA-based thermosensor-4 BBa_K2541304 Cold-inducible RNA-based thermosensor-5 BBa_K2541305 Cold-inducible RNA-based thermosensor-6 BBa_K2541306 Cold-inducible RNA-based thermosensor-7 BBa_K2541307 Cold-inducible RNA-based thermosensor-8 BBa_K2541308 Cold-repressible RNA-based thermosensor-1 BBa_K2541201 Cold-repressible RNA-based thermosensor-2 BBa_K2541202 Cold-repressible RNA-based thermosensor-3 BBa_K2541203 Cold-repressible RNA-based thermosensor-4 BBa_K2541204 Cold-repressible RNA-based thermosensor-5 BBa_K2541205 Cold-repressible RNA-based thermosensor-6 BBa_K2541206 Cold-repressible RNA-based thermosensor-7 BBa_K2541207 Cold-repressible RNA-based thermosensor-8 BBa_K2541208 Cold-repressible RNA-based thermosensor-9 BBa_K2541209 Cold-repressible RNA-based thermosensor-10 BBa_K2541210 References
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