Overview
This year, our project includes a series of mutually orthogonal serine recombinases and their recognition sites, and the "programmable" effect is achieved through the action of these recombinases. We also include the CRISPRi part as an excellent NOT gate in our system, as well as the quorum sensing part as an excellent AND gate in our system. These components are combined through ingenious design to form a programmable logic gate system. In the future, when researchers need a two-input and one-output logic gate, they do not need to design an additional complex pathway. They can get any required logic gate by using our Lantern.
In addition, we also include components such as the Recombination direction factors (RDF) of A118 recombinase and eMutaT7 polymerase. Based on the reversibility of the recombinase inversing effect, we designed a directed evolution system for serine integrase and took the A118 as an example. This continuous directed evolution system is modular. It only needs to replace the recombinase gene, its corresponding recombination site gene, and the corresponding RDF to change the target of continuous directed evolution. This will be helpful to other researchers who want to conduct directed evolution of serine integrase.
New Basic Parts
| ID | Name | Type | Introduction |
|---|---|---|---|
| BBa_K5276001 | A118 | Coding | A118 is a serine integrase that can inverse or excise the DNA sequence between recognition sites depending on the orientation of its specific recognition site. |
| BBa_K5276002 | Bxb1 | Coding | Bxb1 is a serine integrase that can inverse or excise the DNA sequence between recognition sites depending on the orientation of its specific recognition site. |
| BBa_K5276003 | PhiC31 | Coding | PhiC31 is a serine integrase that can inverse or excise the DNA sequence between recognition sites depending on the orientation of its specific recognition site. |
| BBa_K5276004 | Gp44 | Coding | Bacteriophages initially used recombination direction factors (RDF) to cut their own genes out of host DNA to form progeny phages. RDF can control the activity of recombinases to promote specific recombination reaction directions. Research indicates that the Gp44 protein(Mandali et al.,2017), as a recombination direction factor, can enhance the excision or inversion reaction of A118 between attL and attR sites. |
| BBa_K5276023 | TP901 | Coding | TP901 is a serine integrase that can inverse or excise the DNA sequence between recognition
sites depending on the orientation of its specific recognition site. We have improved TP901 and created a new serial number (BBa_K5276023) to store the updated TP901. |
| BBa_K5276010 | eMutaT7 | Coding | MutaT7, initially a fusion of cytosine deaminase (AID) and T7 RNA polymerase (T7RNAP), enables mutations from dC→dT and deoxyguanosine (dG) to deoxyadenosine (dA) (caused by dC→dT mutations on the complementary strand). On this basis, when combined with adenosine deaminase, A→G and T→C mutations can be achieved (A:T→G:C). The enzyme, enhanced for mutagenic activity, is called eMutaT7(Park et al., 2021). |
| BBa_K5276005 | sgRNA_J23111 | RNA | This single guide RNA can be used to inhibit J23111 promoter together with dead cas9(BBa_K3168000). |
| BBa_K5276006 | sgRNA_Trc | RNA | This single guide RNA can be used to inhibit trc promoter together with dead cas9(BBa_K3168000). |
| BBa_K5276011 | TP901B-TC | DNA | This is a specific recognition site for recombinase TP901. It pairs with TP901P-TC(BBa_K5276012). TP901 can catalyze inversion when the sites are anti-aligned or excision when the sites are aligned. |
| BBa_K5276012 | TP901P-TC | DNA | This is a specific recognition site for recombinase TP901. It pairs with TP901B-TC(BBa_K5276011). TP901 can catalyze inversion when the sites are anti-aligned or excision when the sites are aligned. |
| BBa_K5276013 | TP901B-AG | DNA | This is a specific recognition site for recombinase TP901. It pairs with TP901P-AG(BBa_K5276014). TP901 can catalyze inversion when the sites are anti-aligned or excision when the sites are aligned. |
| BBa_K5276014 | TP901P-AG | DNA | This is a specific recognition site for recombinase TP901. It pairs with TP901B-AG(BBa_K5276013). TP901 can catalyze inversion when the sites are anti-aligned or excision when the sites are aligned. |
| BBa_K5276015 | Bxb1B-GT | DNA | This is a specific recognition site for recombinase Bxb1. It pairs with Bxb1P-GT (BBa_K5276016). Bxb1 can catalyze inversion when the sites are anti-aligned or excision when the sites are aligned. |
| BBa_K5276016 | Bxb1P-GT | DNA | This is a specific recognition site for recombinase Bxb1. It pairs with Bxb1B-GT (BBa_K5276015). Bxb1 can catalyze inversion when the sites are anti-aligned or excision when the sites are aligned. |
| BBa_K5276017 | A118B-AA | DNA | This is a specific recognition site for recombinase A118. It pairs with A118P-AA (BBa_K5276018). A118 can catalyze inversion when the sites are anti-aligned or excision when the sites are aligned. |
| BBa_K5276018 | A118P-AA | DNA | This is a specific recognition site for recombinase A118. It pairs with A118B-AA (BBa_K5276017). A118 can catalyze inversion when the sites are anti-aligned or excision when the sites are aligned. |
| BBa_K5276019 | A118B-GG | DNA | This is a specific recognition site for recombinase A118. It pairs with A118P-GG (BBa_K5276020). A118 can catalyze inversion when the sites are anti-aligned or excision when the sites are aligned. |
| BBa_K5276020 | A118P-GG | DNA | This is a specific recognition site for recombinase A118. It pairs with A118B-GG (BBa_K5276019). A118 can catalyze inversion when the sites are anti-aligned or excision when the sites are aligned. |
| BBa_K5276021 | phiC31B-AA | DNA | This is a specific recognition site for recombinase phiC31. It pairs with phiC31P-AA (BBa_K5276022). phiC31 can catalyze inversion when the sites are anti-aligned or excision when the sites are aligned. |
| BBa_K5276022 | phiC31P-AA | DNA | This is a specific recognition site for recombinase phiC31. It pairs with phiC31B-AA (BBa_K5276021). phiC31 can catalyze inversion when the sites are anti-aligned or excision when the sites are aligned. |
Basic parts from previous teams
| ID | Name | Type | Introduction |
|---|---|---|---|
| BBa_K1132010 | TP901 | Coding | TP901 is a serine integrase that can inverse or excise the DNA sequence between recognition sites depending on the orientation of its specific recognition site. |
| BBa_K137007 | FimE | Coding | Fime is an integrase that catalyzes the inversing or excision of DNA sequences between its recognition sites. |
| BBa_K1021005 | Cre | Coding | The Cre recombinase can be used for site-specific recombination of constructs flanked by lox sites (BBa_J61046). The Cre-Lox recombination system can be used for gene manipulation and as a biosafety mechanism. As a biosafety mechanism, its action is to remove engineered genes flanked by lox sequences. |
| BBa_K3168000 | dcas9 | Coding | Cas9 (CRISPR associated protein 9) is an RNA-guided DNA endonuclease which is part of the CRISPR-immune system of bacteria such as Streptococcus pyogenes. This endonuclease is used a lot in research to facilitate efficient genome engineering (Ran, 2013). Cas9 can target any sequence by simply specifying a 20 nucleotide targeting sequencing within the guide RNA. dCas9 is a 'dead' variant of Cas9, because it does not have its endonuclease activity. This means that dCas9 binds dsDNA but does not cut the DNA, because of mutations in the RuvC1 and HNH nuclease domains (Park, 2017). |
| BBa_K2152003 | Phi X 174 lysis gene E | Coding | Lysis protein E spanning the inner and outer membrane of the bacterial, leading to low local degradations of peptidoglycan, allow the release of cytoplasmic content. |
| BBa_K1680005 | loxp | DNA | This part contains the WT loxP site which is recognised by Cre recombinase (e.g. BBa_K1680007), in forward orientation. |
| BBa_K3963005 | trc promoter | Regulatory | trc promoter is a strong Escherichia coli promoter and a hybrid between the trp and lac UV5 promoters. It was used additionally for Acinetobacter baylyi ADP1. |
| BBa_K914003 | rha promoter | Regulatory | L-rhamnose-inducible promoter is capable of high-level recombinant protein expression in the presence of L-rhamnose, it is also tightly regulated in the absence of L-rhamnose by the addition of D-glucose. |
| BBa_B0015 | double terminator | Terminator | A two-way terminator. |
| BBa_K2598002 | ECK120033737 | Terminator | A strong synthetic terminator. |
New Composite Parts
| ID | Name | Introduction |
|---|---|---|
| BBa_K5276007 | cymR | The cumate inducible promoter, consists of Psyn2 flanked by two CuO operator sequences; expression of a high-GC-content codon-optimized gene for the CymR repressor (cymR) is driven divergently by a modified bla promoter (Pbla-mut1T) in which the 35 box has been changed to TTGACA and the 10 box to TA |
| BBa_K5276008 | xylO | This system was constructed by combining the xylose repressor gene xylR, its operator sequence xylO, and the strong constitutive promoter PxylAB, the divergent promoter to drive the expression of downstream gene. The XylR is expressed constitutively from PxylR. It binds to the XylR operator and blocks downstream gene expression from the PxylAB promoter in the absence of xylose. XylR is released from the operator with xylose, initiating the downstream gene expression. |
| BBa_K5276009 | DAPG | In the absence of DAPG, the TetR-like repressor protein PhlF binds as a dimer to the phlO operator site in the promoter upstream of the reporter gene. As bioavailable DAPG diffuses into the cytoplasm and binds to PhlF, the repression on the target PphlF promoter is relieved. |
| BBa_K5276024 | Tangram | Tangram is an expression unit composed of four different inducible promoters(BAD promoter,LeteO promoter,Phlf promoter,xylO promoter) , each expressing a different recombinant enzyme(tp901,Bxb1,A118,PhiC31). |
| BBa_K5276025 | Register 0 | Register 0 is a regulation part that can be used to verify the feasibility of our Register system; it can verify whether two or more promoters can work together and whether it is effective to take regulatory proteins like LacI apart from promoters like Plac. |
| BBa_K5276026 | Register A | Register A is a unit of expression consisting of four promoters(constant promoter J23111, trc promoter, and inducible promoter lac operon, Rha promoter) with different directions, Three pairs of DNA recognition sites(TP901B-TC_TP901P-TC, Bxb1B-GT_Bxb1P-GT, TP901B-AG_TP901P-AG), a Ribosome binding site, and LuxI. |
| BBa_K5276027 | Register B | Register B is a unit of expression consisting of four promoters(constant promoter J23111, trc promoter, and inducible promoter lac operon, Rha promoter) with different directions, Three pairs of DNA recognition sites(A118B-AA_A118P-AA, PhiC31B-AA_PhiC31P-AA, A118B-GG_A118P-GG), a Ribosome binding site, and LuxR. |
| BBa_K5276028 | CRISPRi | The CRISPRi system primarily consists of three promoters (the constitutive promoter J23111, the inducible lac operon promoter, and the Rha promoter) that respectively express dCas9 protein, sgRNA1, and sgRNA2. Additionally, it includes a gRNA scaffold, a T7 terminator, and a dbl terminator. By combining the CRISPRi system, the recombinase expression system (Tangram section) and the sequences above, we can achieve the transformation of logic gates by adding recombinant enzymes in different orders. |
| BBa_K5276029 | Patch | Patch system uses Cre/loxP system and fimE inversion system. This constitutes the Patch portion of Register A. Similarly, in the Register B pathway, we incorporated the PobR promoter used by the 2023 UCAS-China team and the fimE inversion system from Escherichia coli (Ham et al., 2006) to complement the expression of the LuxR gene. |
| BBa_K5276030 | J23119_Promoter-a118-GFP | One of the three plasmids for directed evolution. A constitutive promoter, J23119, is used to express the inverted GFP gene. |
| BBa_K5276031 | PBAD_Promoter-eMutaT7 | One of the three plasmids for directed evolution. The eMutaT7 enzyme is expressed under the control of the PBAD promoter, which is induced by arabinose. |
| BBa_K5276032 | T7_Promoter-lacO-tetR-A118-lacI-Gp44 | One of the three plasmids for directed evolution. Recombination direction factors (RDF) can control the activity of integrases to promote specific recombination reaction directions. We designed a bistable switch system focused on regulating the expression of the A118 integrase and the Gp44 protein. The "Turn over" switch is controlled by the lactose operon, while the "Turn back" switch is regulated by the tetR protein. |
| BBa_K5276033 | Selection plasmid of traditional mutagenesis | We designed expression plasmid and selection plasmid. The expression plasmid induces recombinant enzyme expression with IPTG. We incorporated mixed NNK mutation sequence primers into the PCR system to generate a library of various mutants. The mutation and screening plasmids were then introduced into E. coli. |
| BBa_K5276034 | Expression plasmid of traditional mutagenesis | We designed expression plasmid and selection plasmid. The selection plasmid uses a phage lysis gene φX174 E. We incorporated mixed NNK mutation sequence primers into the PCR system to generate a library of various mutants. The mutation and screening plasmids were then introduced into E. coli. |
Best Part Collection
This year, our project includes
- six recombinases and their corresponding eight pairs of recombination sites
- some inducible promoters and constitutive promoters
- components related to the CRISPRi system and the quorum sensing system
- eMutaT7 and Gp44
By integrating these components, we have obtained a "Tangram-Register-Output" system.
"Tangram" consists of six recombinase genes positioned behind six inducible promoters, enabling us to control the expression of different recombinases by adding specific chemical inducers. These recombinases can invert or excise genes between their respective recognition sites. We create our core "Register" component—comprising RA, RB, and a patch—by combining various recombination sites with constitutive and inducible promoters. The pathways diverge based on the recombinase inputs, allowing for the formation of different logical relationships. Ultimately, the signals from multiple pathways in the "Register" are integrated and output through the quorum sensing element in the "Output" section.
Furthermore, by combining eMutaT7 and Gp44, we created a continuous directed evolution system for serine integrases and used the A118 integrase as an example. This system is highly substitutable, and we can conduct continuous directed evolution on any serine integrase by simply changing the corresponding recombination direction factors (RDF) and recombination sites. We hope that our continuous directed evolution system can be universally applicable to any member of the serine integrase family, thereby improving the entire serine integrase family and providing subsequent iGEMers with more efficient serine integrases.
