Team:UNESP Brazil/Engineering

Project


DESIGN

     We made the design to optimize the project's innovation pillars, the autonomous and temporal regulation of the system. Thus, we divided the design into three modules: control, message, and response. Each module is responsible for regulating the circuit, carrying out the progression and continuity of the code, and issuing the signals, respectively.

     The control module consists of 3 elements: the TetR repressor protein, controlled by the J23100 promoter (constitutive and strong of the Anderson family for E. coli); SpdCas9 fused to bacteriophage T4 anti-sigma 70 protein, AsiA, controlled by the Tet promoter (regulated by TetR); and gRNA-S, controlled by the T7-lacO promoter (related to T7 RNA polymerase and with a regulatory site for the LacI protein). To better understand and visualize the control module, you can join the previous information with the image below.

Image 1. Graphical representation of the control module. (a) Constitutive PJ23117 promoter controlling the synthesis of the TetR repressor protein. (b) TetR protein negatively regulating the Ptet promoter, which regulates the synthesis of SpdCas9 protein fused to the AsiA activator protein. In the presence of anhydrotetracycline (aTc), the promoter repression does not occur due to the formation of TetR-aTc complex. (c) LacI protein negatively controlling the PT7-lacO promoter, through interaction with the lac operator site, which controls the transcription of the gRNA-S.

     After the start of gRNA-S transcription, Message Module will start. The Message Module is composed of seven blocks, composed of the following parts: a target site (Sa and 3a to 7a) of activation for the SpdCas9_AsiA_gRNA complex, the PJ23117 promoter (weak promoter, constitutive of the Anderson family for E. coli), the ribozyme RiboJ, gRNAs 0 and 1 and gRNAs 3 to 7. The system will work through the transcription with time spacing of each of the blocks. The 0 and 1 gRNAs are responsible for guiding the activation complex to the response module and thus transcribing the fluorescent RNAs. On the other hand, gRNAs 3 to 7 have the function of interspersing the synthesis of gRNAs 0 and 1 because they guide the activation complex through parts of the Message Module (3 to 7), producing a temporal variation in the transcription of gRNAs 0 and 1, hence a variation in fluorescent RNAs. In addition, the gRNAs in this module contain RiboJ, a self-cleaving ribozyme that will separate the two gRNAs after transcription, in addition to increasing their stability.

     The last division of our system corresponds to the Response Module. This circuit module contains the parts necessary to create the binary code message through fluorescence. For this, there are two parts composed of the RNAs Broccoli and Corn genes, which will be the numbers 0 and 1 of the code, controlled by the PJ23117 promoter. In addition, there are four target sites: 0i, 0a, 1i, and 1a. The "a" sites are for activating gene transcription, and the "i" sites are for repressing the gene. Thus, when gRNA-0 reaches a minimum concentration in the system, the transcription of RNA Broccoli and repression of RNA Corn will occur. When the system transcribes the gRNA-1, the opposite happens. With this, it will be possible to differentiate the message through the time needed to change the concentration of gRNAs 0 and 1.

    


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BUILD

     The Benchling platform was used to design all project parts. The table below show all the parts synthesized in gBlocks, by IDT.

The parts have restriction sites following the BioBrick and Golden Gate cloning patterns, to be later joined in plasmids pSB1A3 (Control Module) and pSB1K3 (Message and Answer Module). The dcas9 and tetR genes were amplified by PCR from a laboratory plasmid. The other sequences were synthesized by IDT in gBlocks. The gBlocks containing the gRNA-S and all other gRNAs (3 to 8) contain the BsaI enzyme site to be joined by Golden Gate. The gBlock of the target-0a_PJ23117_target-1i_broccoli part has been split into two parts and has been joined by Golden Gate. The union of non-equal parts was also performed:

PT7-lacO_gRNA-0+target-1a_PJ23117_target-0i_corn

PT7-lacO_RiboJ_gRNA-0+target-1a_PJ23117_target-0i_corn

PT7-lacO_gRNA-1+target-1a_PJ23117_target-0i_corn

PT7-lacO_RiboJ_gRNA-1+target-1a_PJ23117_target-0i_corn

     These parts will work as a proof of concept for the system, as they do not have the complete message, only a Corn activation gRNA (1) and a repression gRNA (0).

     Initially, parts 1-4 (pSB1K3) and tetR-dcas9 (pSB1A3) were cloned in E. coli Top10, as this strain has greater replication capacity. These clonings were confirmed by digestion and agarose gel electrophoresis, in addition to Sanger sequencing. To prove the functionality of the genetic circuit the parts will be cloned into BL21 since this bacterium has T7 RNA polymerase.


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TEST

     Tests were performed in silico with iBioSim 3.0 software. Click Here for more data.


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LEARN

     As engineering success we were able to ligate gBlocks 8.1 and 8.2 through GoldenGate with the BsaI enzyme. The image below shows the PCR extension of the target-0a_J23117_target-1i_broccoli sequence, confirming the success of GoldenGate.

Image 4. Electrophoresis gel of target0a_PJ23117_target1i_broccoli after Golden Gate reaction.

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