1. Construction of SSR Collection

The expression of genes is regulated by the promoter, secondary structures of 5’-UTR, the strength of RBS and even up elements. Meanwhile, insulators are powerful tools for buffering noise so as to achieve a controllable expression in the view of synthetic biology. Furthermore, inducible promoters play an essential role in the work of complex gene circuits because the condition of chassis organism can be controlled "on" or "off" as we designed and the whole burden will also decrease.

As a chassis for synthetic biology, a matched complex toolkit which makes the chassis work as we designed is a focused field, meaning that parts with high efficiency and various functions should be developed. Parts that have been characterized in Vibrio.natriegens is limited and choice of inducible promoters is narrow, however, these impede the extensive use and of Vibirio.natriegens in synthetic biology.

Here, we built a golden-gate-based collection called SSR (strick and subtle regulation) collection containing more natural or artificially designed parts, efficient inducible promoters and a tunable ORI as well .

Team Marburg has built a collection containing the promoter, RBS, terminator, tags, and different ORIs. Promoters in this collection have a high homogeneity, however, which may cause homologous recombination in the V.n due to its rapid growth rate. What’s more, the copy number spectrum of ORIs is narrow and regulation methods are lack due to the poor measurement of inducible promoters.

This year, taking up-elements, insulator, special 5’-UTR, inducible promoters, artificially designed promoters, and a tunable ORI into consideration, we expended the Marburg collection, hoping to develop a collection for strict and subtle regulation (SSR collection).

1. Up-elements: In SSR collection, the up-elements is rich in PAM 5’-TBN-3’ which is compatible with CasΦ-2 (CasΦ for short below) , so the expression of genes in our circuit can be regulated by CRISPRa.

2. Insulators: The insulator is a type of self-cleavage ribozyme that could cut itself as quickly as transcription reaches the 5’ of insulator. In this iGEM project, we use RiboJ widely.

3. Twelve generators based on Marionette collection: All of them are from E.coli marionette collection, and most of them show fantasy cooperative in E.coli. Here, we introduced and modified them in Vibirio.natriegens.

4. Artificial designed promoters: All of them are from E.coli marionette collection, and most of them show fantasy cooperative in E.coli. Here, we introduced and modified them in Vibirio.natriegens.

5. Tunable ORI:After characterizing basic ORIs in Vibirio.natriegens, we found ColE1 performed well, so we engineering it into Tet-ColE1 so as to control the copy number in a wide range

It should be mentioned that the construction of basic part collection needs a mass of labor and reagent, and for the most of research groups especially iGEM teams, expenditure and vim are limited. Here, we develop a strategy of collection construction at a low cost.

Take the SSR collection as an example, most of our basic parts is a short DNA sequence almost 35bp to 80bp, and primers < 59nt are cheapest in China, so we constructed basic parts as Figure 1

Figure 1. Work flow of duilding level0 parts in different length

As shown in Figure 1.A, for those DNA fragments with a sequence not beyond 51 bp, we choose to ligate them into our pre-made backbone with two stick-ends by T4 ligase. So we just need to anneal a pair of primer in the thermocycler and then ligate it with the backbone before the transformation. For some especially short sequences, such as RBS, we will use the strategy as Figure 1.B and obtain the whole DNA sequence by annealing. For longer sequences, PCR without any other templates will be applied.

2. Construction and Optimization of CRISPRa

In prokaryote, most of the CRISPRa strategies are based on the activation domains (AD) which could recruit components of RNAp, so as to direct the work of holoenzyme. Until now, three manners of recruiting ADs nearby promoter have been demonstrated successful gene activation as shown in Figure 2. Most of related CRISPRa research directly fused AD with dCas9 or dCas12a and usually achieve the best efficiency. Another popular manner is applying RNA-binding protein and special RNA adapter such as MCP-MS2 and γN22plus-BoxB. This manner could achieve the same activation level compared with direct fusion. In a latest research, SYNZIP was used to recruit AD noncovalently and also achieve a high activation.Although they achieve CRISPRa in different manner, the key princinple is drawing up AD with dCas which is bound nearly promoter so as to recruit RNAp. Based on this, we assumed that intein can also help dCas to achieve CRISPRa,

Figure 2. Work flow of duilding level0 parts in different length

The function of σ70-based CRISPRa is also regulated by other factors. Distance between PAM site and TSS (transcription start site) could influence the efficiency by changing the relative distance and conformation of CRISPRa construction to the promoter. Interestingly, the gene expression seems like be regulated by different saturated levels in different species, meaning that the activation efficiency of stronger promoter is usually lower than weaker ones.

Based on theories menthioned above, we planed to apply DBTL to choose the best activation domains and the appropirate construction under the help of protein structure model.

It's mentioned that Vibrio natriegens is sensitive to the burden caused by exogenous genes, which means the complex gene circuit based on regulator proteins such as TetR, CI and even dCas9 could cause high burden and their function may be loosed. These years, two miniature Cas protein, CasΦ and AsCas12f1 have been discovered, so we chose them as a Cas part to accomplish CRISPRa.

Figure 3. Work flow of duilding level0 parts in different length

When it comes about the activation mechanism, the recruitment of RNAp is the princinple of σ70-based CRISPRa, so the capacity of AD to recruit RNAp should be evaluated so as to choose the best AD for CRISPRa. Until now, E.c SoxS, P.a α-NTD, E.c α-NTD, E.c omega, E.c σ70 have been used to fuse with dCas protein and accomplish CRISPRa. We chose several domains from them and then colone homology genes from Vibrio.natriegens's genome in order to test their functions with the help of model analysis, and engineered the whole CRISPRa construction by location and linker optimization.

3. Construction of logical gene circuits by dCasΦ

To further use dCasΦ in the construction of complex regulatory networks, we explored the feasibility of dCasΦ for the construction of logical gene circuits. Compared to protein regulators, CRISPRi is higher programmability, orthogonality, predictability and lower burden due to its RNA-guided nature[22].Although the lack of cooperativity was previously considered a potential drawback of constructing CRISPRi-based dynamic and multistable circuits.The unspecific binding in CRISPRi make it possible to establish multistability[23].

We designed a Toggle Switch(TS) based on dCasΦ and selected the highly orthogonal fluorescent proteins sfGFP and mScarlet-I as reporter genes. Two nodes of TS produce crRNAs that repress each other, resulting in two non-concurrent stable states(Figure 4a): high expression of sfGFP while low expression of mScarlet-I, high expression of mScarlet-I while low expression of sfGFP. These two states can be regulated by a controller plasmid, where the addition of Ara or aTc triggers the expression of crRNAsfGFP or crRNAmSarlet-I to switch states(Figure 4b).

Figure 4. a Topology of the bistable TS. b. Gene circuit of the TS. In addition, Reporter genes,AraC, TetR and dCasΦ are constitutively expressed. Bent arrows: promoters; circles: Ribo10; and T-s: transcriptional terminators. 

For the reporter plasmid, to prevent mRNA context-dependency and provide transcriptional insulation within TUs, we employed RiboJ10[24] processing to release parts that are transcribed together in the same mRNA molecule but need to act independently once transcribed—e.g., crRNAs that need to bind dCasΦ for function, and fluorescent reporter transcripts that have to be translated. The recognition sites of BsaI and BsmBI was added downstream of the two crRNA-scaffolds respectively in anticipation of a rapid spacer substitution based on Golden Gate Assembly(Figure 5).

Figure 5. Spacer substitution steps (ORF of mScarlet-I as an example). Bent arrows: promoters; circles: Ribo10; crosses: BsmBI cleavage sites and T-s: transcriptional terminators.  


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