Parts Library
Design considerations
The directed evolution system TRACE was first reported by the Chen Fei research group at MIT. The key plasmid, pEditor (BBa_K5005003), used in our experiments, was provided by Professor Yu Yang from the Institute of Biophysics, Chinese Academy of Sciences. Firstly, we verified the feasibility of our directed evolution system (TRACE) using a customized EGFP plasmid. In principle, we mutated the 199th base of the EGFP gene (BBa_K5005001) from T-A to C-G, converting tyrosine to histidine and changing the fluorescence color from green to blue (BBa_K5005000). Therefore, if the TRACE system is feasible, we can mutate the BFP gene obtained from site-directed mutagenesis back to the EGFP gene, achieving a change in cell fluorescence from blue to green. In specific steps, (1) we first constructed an empty plasmid, pTarget (BBa_K5005014): the target plasmid contains a T7 promoter downstream of the CMV promoter from the pCDH vector; (2) Then, we mutated the reporter gene from EGFP (BBa_K5005001) to BFP (BBa_K5005000); (3) Through Biobricks provided by iGEM and homologous recombination, we got pTarget-BFP (BBa_K5005002).
BBa_K5005000(BFP)
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Reporter
BFP
Design considerations: In target plasmid(BBa_K5005014), BFP will be cloned to the downstream of the T7 promoter (derived from site-directed mutagenesis of EGFP), expressing the target gene BFP (blue fluorescence), which could potentially be evolved back into EGFP by Editor.
By transforming the EGFP (BBa_K5005001), the target BFP fragment is obtained.
Description: EBFP is utilized to verify the feasibility of the TRACE system. EBFP was derived from site-directed mutagenesis of EGFP, which has the potential to be evolved back into EGFP by the Editor, can be utilized to verify the feasibility of the TRACE system.
Length: 720 bp
BBa_K5005001(EGFP)
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Reporter
EGFP
Design considerations:
Target plasmid with EGFP located downstream of the T7 promoter. EGFP can be converted into BFP through site-directed mutagenesis, which changes one amino acid. This eGFP serves as a template for our transformation, using point mutations to mutate it into the BFP we need to change green fluorescence into blue fluorescence.
Description: The BFP protein is located downstream of the T7 promoter.
Length: 720 bp
BBa_K5005002(pTarget)
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Plasmid_Backbone
The BFP protein is located downstream of the T7 promoter.
Design considerations: The Target plasmid expresses the target gene BFP (blue fluorescence), derived from site-directed mutagenesis of EGFP, which has the potential to be evolved back into EGFP by the Editor. BFP is positioned downstream of the T7 promoter, enabling specific editing of EBFP by the Editor. If the TRACE system operates effectively, it is feasible to observe cells where blue fluorescence is converted into green fluorescence.
Description: BFP is located downstream of the T7 promoter (derived from site-directed mutagenesis of EGFP), expressing the target gene BFP (blue fluorescence), which could potentially be evolved back into EGFP by Editor(BBa_K5005003).
Length: 8023 bp
BBa_K5005003(AID-T7 RNAP-UGI)
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Coding
Expresses editor under dox induction.
Design considerations: Editor plasmid, expresses editor under doxycycline induction, which can edit genes downstream of the T7 promoter on the pTarget plasmids.
Description: BFP is located downstream of the T7 promoter (derived from site-directed mutagenesis of EGFP), expressing the target gene BFP (blue fluorescence), which could potentially be evolved back into EGFP by Editor(BBa_K5005003).
Length: 3576 bp
BBa_K5005004(NAMPT)
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Coding
AS PCR template for NAMPT fragment cloning.
Design considerations: This part contains NAMPT Coding Sequence. Through subsequent editing and transformation of the gene, it will help increase NAD+ biosynthesis. NAMPT was cloned using PCR. Homologous arms play a crucial role in the Gibson assembly for connecting NAMPT with the vector.
Description: NAMPT is a key rate-limiting enzyme for NAD+ biosynthesis, and it is the gene we aim to evolve in our project. We obtained the gene sequence of NAMPT from published literature and cloned it into a vector that can be expressed in mammalian cells through molecular cloning.
Length: 1476 bp
BBa_K5005005(pCDH-CMV-T7-NAMPT)
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Plasmid
Primordial plasmid before point mutation.
Design considerations: NAMPT was cloned using PCR. Homologous arms play a crucial role in the Gibson assembly for connecting NAMPT with the vector. After obtaining the original pTarget-NAMPT plasmid, NAMPT will undergo site-directed mutagenesis at specific points to validate the impact of different mutations on NAMPT enzyme activity. Additionally, in the pTarget-NAMPT plasmid, NAMPT will be positioned downstream of the T7 promoter for directed evolution experiments on NAMPT. Here, you can clearly know that NAMPT is located downstream of the T7 promoter and can be edited by the editor to introduce mutations, resulting in NAMPT variants.
Description: NAMPT (BBa_K5005004) is a key rate-limiting enzyme for NAD+ biosynthesis, and it is the gene we aim to evolve in our project. We obtained the gene sequence of NAMPT from published literature and cloned it into pTarget plasmid (BBa_K5005002) that can be expressed in mammalian cells through molecular cloning. This new NAMPT-expressing vector is also compatible with the pEditor system (BBa_K5005003) for directed evolution.
Length: 8732 bp
BBa_K5005006(NAMPT-V221G)
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Coding
The product of NAMPT point mutation - V221G.
Design considerations: To verify the point mutations during the screening process.
Description: The 221st amino acid Valine in the NAMPT protein is its active site. In the preliminary evolution experiments done by our advisors, they found that this site can be mutated into Glycine. Due to the time constraints of the iGEM competition, we were unable to complete the entire process of NAMPT evolution experiments. However, we have successfully constructed this mutated plasmid, which can be used for subsequent enzyme activity validation.
Length: 1476 bp
BBa_K5005007(NAMPT-V221A)
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Coding
The product of NAMPT point mutation - V221A.
Design considerations: To verify the point mutation in the patent CN 113106080 A.
Description: Through patent search, we discovered that some sites on the NAMPT protein have been confirmed through experiments such as prokaryotic expression, protein purification, and enzyme activity assays to enhance the enzymatic activity of NAMPT. Due to the time constraints of the iGEM competition, we were unable to complete the entire process of NAMPT evolution experiments. However, we have successfully constructed these mutated plasmids, which can be used for subsequent enzyme activity validation. As reported by the patent, the Val to Ala mutation increases enzyme activity by 337%.
Length: 1476 bp
BBa_K5005008(NAMPT-D354E)
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Coding
The product of NAMPT point mutation - D354E.
Design considerations: To verify the point mutation in the patent CN 108026517 B.
Description: Through patent search, we discovered that some sites on the NAMPT protein have been confirmed through experiments such as prokaryotic expression, protein purification, and enzyme activity assays to enhance the enzymatic activity of NAMPT. Due to the time constraints of the iGEM competition, we were unable to complete the entire process of NAMPT evolution experiments. However, we have successfully constructed these mutated plasmids, which can be used for subsequent enzyme activity validation. As reported by the patent, the Asp to Glu mutation increases enzyme activity by 416%.
Length: 1476 bp
BBa_K5005009(NAMPT-G384C)
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Coding
The product of NAMPT point mutation - G384C.
Design considerations: Through patent search, we discovered that some sites on the NAMPT protein have been confirmed through experiments such as prokaryotic expression, protein purification, and enzyme activity assays to enhance the enzymatic activity of NAMPT. Due to the time constraints of the iGEM competition, we were unable to complete the entire process of NAMPT evolution experiments. However, we have successfully constructed these mutated plasmids, which can be used for subsequent enzyme activity validation. As reported by the patent, the Gly to Cys mutation combined with two other mutations mentioned in the patent increases enzyme activity by 35.6%.
Length: 1476 bp
BBa_K5005010(NAMPT-L386P)
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Coding
The product of NAMPT point mutation - L386P.
Design considerations: To verify the point mutation in the patent CN 113106080 B.
Description: Through patent search, we discovered that some sites on the NAMPT protein have been confirmed through experiments such as prokaryotic expression, protein purification, and enzyme activity assays to enhance the enzymatic activity of NAMPT. Due to the time constraints of the iGEM competition, we were unable to complete the entire process of NAMPT evolution experiments. However, we have successfully constructed these mutated plasmids, which can be used for subsequent enzyme activity validation. As reported by the patent, the Leu to Pro mutation increases enzyme activity by 551%.
Length: 1476 bp
BBa_K5005011(pCDH-CMV-mCherry-FiNad)
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Plasmid
Express NAD+ biosensor- FiNad.
Design considerations: The FiNad biosensor is employed as an NAD+ biosensor for real-time monitoring of cellular NAD+ level fluctuations.
Description:
Length: 8342 bp
BBa_K5005013(FiNad)
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Coding
It is a genetically encoded NAD+ biosensor.
Design considerations: The FiNad biosensor is employed as an NAD+ biosensor for real-time monitoring of cellular NAD+ level fluctuations. By synthesizing the mCherry-FiNad nucleotide sequence, it can be ingeniously ligated to the pCDH vector for achieving stable expression in mammalian cells. This allows real-time monitoring of dynamic NAD+ fluctuations in live cells.
Description: The FiNad biosensor is employed as an NAD+ biosensor for real-time monitoring of cellular NAD+ level fluctuations. By synthesizing the mCherry-FiNad nucleotide sequence, it can be ingeniously ligated to the pCDH vector for achieving stable expression in mammalian cells. This allows real-time monitoring of dynamic NAD+ fluctuations in live cells.
Length: 2099 bp
BBa_K5005014(pTarget)
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Plasmid
pTarget
Design considerations: Target plasmid contains a T7 promoter downstream of the CMV promoter. The target gene will be inserted into the multiple cloning site downstream of the T7 promoter. This T7 promoter enables the editor to perform gene editing specifically on the target gene inserted after the T7 promoter.
Description: pTarget
Length: 7263 bp
Reference
1. Zhu, X., Liu, H., Chen, L., Wu, C., Liu, X., Cang, Y., Jiang, B., Yang, X., & Fan, G. (2022). Addressing the Enzyme-independent tumor-promoting function of NAMPT via PROTAC-mediated degradation. Cell chemical biology, 29(11), 1616-1629.e12. https://doi.org/10.1016/j.chembiol.2022.10.007.
2. Chen, H., Liu, S., Padula, S., Lesman, D., Griswold, K., Lin, A., Zhao, T., Marshall, J. L., & Chen, F. (2020). Efficient, continuous mutagenesis in human cells using a pseudo-random DNA editor. Nature biotechnology, 38(2), 165-168. https://doi.org/10.1038/s41587-019-0331-8.
3. Zou, Y., Wang, A., Huang, L., Zhu, X., Hu, Q., Zhang, Y., Chen, X., Li, F., Wang, Q., Wang, H., Liu, R., Zuo, F., Li, T., Yao, J., Qian, Y., Shi, M., Yue, X., Chen, W., Zhang, Z., Wang, C., … Zhao, Y. (2020). Illuminating NAD+ Metabolism in Live Cells and In Vivo Using a Genetically Encoded Fluorescent Sensor. Developmental cell, 53(2), 240-252.e7. https://doi.org/10.1016/j.devcel.2020.02.017.