Team:SCUT China/Design

Ruby - Responsive Corporate Tempalte

Balancing gene expression is critical when optimizing genetic systems. Typically, this requires library construction to vary the genetic parts controlling each gene, which can be expensive and time consuming. With these realities in mind, we have adopted the engineered philosophy of synthetic biology to design our experiments：decoupling and standardization[1] .

 

Decoupling


Decoupling is the process of decomposing a complex problem into many relatively simple problems that can be dealt with independently and finally integrating them into a unified whole with specific functions. In our project design, the difficulty lies in how to find the optimal promoter regulatory combination. The ideal approach is to create a library in which the expression levels of multiple genes can be adjusted simultaneously without the need to rebuild a library for each system.

To achieve this regulation approach that variation can be achieved with a separate library, built once, that contains regulators expressed at different levels, we need to design this system in which each regulator could control a different gene so that changes in regulator expression lead to changes in the target gene.

This approach requires regulators that are[2]:

  • √     Orthogonal so that each regulator only controls its target.
  • √     Wide in dynamic range to be able to sweep across expression levels.
  • √     Programmable so that the regulator can be targeted to different genes.
  • √     Non-toxic so that the regulators themselves do not influence the system.

After extensive literature review, we chose coupling the Toehold Switch and T7 promoters as the composite foundation elements for the VerProS pool on account of meeting the standards above.

 

T7 promoters library -- Wide in dynamic range to be able to sweep across expression levels.

The T7 promoter is recognized by T7 RNA polymerase. Since T7 RNA polymerase is a very powerful RNA polymerase, it can synthesize mRNA several times more efficiently than E. coli RNA polymerase, and stops transcription less frequently, so it is enough to stop the background expression of E. coli [2]. Therefore, we selected ten T7 promoters of different strengths from the current T7 promoter library, covering the strongest to the weakest. And We have upload these sequences as our basic parts. (BBa_K3100001 to BBa_K3100010) In order to achieve a larger scale and more accurate regulation range, we have improved T7 promoters with different strength providing more options for the precise regulation. (BBa_K3100022-BBa_K3100031) 


Fig 1. Site-saturation mutagenesis was conducted to obtain the T7 mutants

 

Toehold Switch -- Orthogonal so that each regulator only controls its target.

Toehold Switch systems are composed of two RNA strands referred to as the switch and trigger. The switch RNA contains the coding sequence of the gene being regulated. Upstream of this coding sequence is a hairpin-based processing module containing both a strong ribosome binding site and a start codon that is followed by a common 21-nt linker sequence coding for low molecular weight amino acids added to the N-terminus of the gene of interest. A single-stranded Toehold sequence at the 5’ end of the hairpin module provides the initial binding site for the trigger RNA strand[3]. This trigger molecule contains an extended single-stranded region that completes a branch migration process with the hairpin to expose the RBS and start codon, thereby initiating translation of the gene of interest. The hairpin processing unit functions as a repressor of translation in the absence of the trigger strand. (BBa_K3100011-BBa_K3100016)


Fig 2. Design schematics of Toehold Switch

 

VerProS System—Upgrade of the Toehold Switch and T7 Promoter Library.

After finding suitable components, we began to think about how to specifically build our library.
First, Toehold Switch is used to separate the T7 promoter from the gene, and a set of dual plasmid system -- transcription vector and working part.

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Fig3. Schematic diagram of VerProS pool

 

When the promoter strength corresponding to trigger is higher, the transcription level of trigger will be improved, which will lead to more Toehold Switch being turned on, and the gene expression level will also be increased. Therefore, the stronger the promoter, the higher the gene expression. Therefore, we can find the optimal gene expression level combination by changing the combination of promoter strength.
Therefore, coupling the Toehold Switch and T7 promoters as the composite foundation elements, variation  of the regulation can be achieved with a separate library. After putting forward our design ideas, we need to clarify our standardized construction process.

 

Standardization


To achieve versatility of the pool, connections between different parts need to be standardized. Only when these standardized specifications are widely adopted can the units designed and constructed by different researchers be guaranteed to match each other. It will be more conducive to accelerate and protect the exchange and use of genetic information of specific biological parts and share. 

The principle of our project cloning strategy is based on the ability of type IIs restriction enzymes to cut outside of their recognition site. Two DNA ends can be designed to be flanked by a type IIs restriction site such that digestion of the fragments removes the enzyme recognition sites and generates ends with complementary 4nt overhangs; such ends can be ligated seamlessly, creating a junction that lacks the original site.[4]

 

VerProS pool -- The dynamic range of control can be increased through standardization

pET30a(+) is selected as the plasmid skeleton of the transcription vector, and the lethal gene ccdB is used to select the right splicing transcription plasmid. 40 plasmids containing one promoter and one Trigger each are constructed by free combination of 10 various promoters with 4 different triggers. Finally, these 40 plasmids are randomly combined with pET30a(+)-ccdB plasmids vector through Golden Gate assembly. These eventually constitute the VerProS pool with a capacity of 10000. And because of standardized recombination sites design, we can easily increase the dynamic range of control and expand the storage capacity of the pool in the future.
(BBa_K3100100-BBa_K3100139)

Fig4. T7 promoter & Trigger DNA composite parts assembly method

 


  • Fig5. Golden Gate Assembly of VerProS Pool

     

    Working part -- The versatility of pool can be achieved through standardization

    The working part carries four key genes in the metabolic process required for regulation and four Toehold Switch sequences corresponding to the Trigger on the transcription plasmid. The recognition and specific recombination sites of the BsaI enzyme were added to both ends of the gene and Toehold Switches. Then the plasmids shown in the following figure were constructed. Finally, Golden Gate assembly was used to construct the plasmids together with the vector plasmid with ccdB into the final working part.



    Fig6. Gibson Assembly or Gene Synthesis for basic parts constructing




    Fig7. Golden Gate Assembly of Acid Tolerant Working Part

     

    Demonstrate -- The feasibility of pool can be tested for acid tolerant

    After sufficient investigation and research, we decided to introduce gadB, gadC, ybaS and katA, which can regulate acid-tolerant genes, into the E. coli MG1655 expression system to complete the relevant test of our VerProS pool. BBa_K3100017-BBa_K3100020, BBa_K3100140

     

    Self-design algorithm -- Help standardize design

    Recombination sites would be chosen on sequences that are identical among all homologues, but different from all other selected sites within the same gene. An algorithm was designed by us to detect the specificity of recombination sites. 
    （Click here to download: Enzyme digestion sites optimization algorithm of Golden Gate Assembly designed by SCUT_China）

     

  • Reference:

    [1] Bhutkar A.(2005). Synthetic biology: navigating the challenges ahead.
    [2] Amar Ghodasara, Christopher A. Voigt, et al. (2017). Balancing gene expression without library construction via a reusable sRNA pool.
    [3] Alexander A. Green, Pamela A. Silver, et al.(2015). Toehold Switches De-Novo-Designed Regulators of Gene Expression
    [4] Carola Engler, Ramona Gruetzner，et al.(2009). Golden Gate Shuffling: A One-Pot DNA Shuffling Method Based on Type IIs Restriction Enzymes.

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