By constructing and assembling various components, our project has successfully built two generations of black box systems. By applying our black box systems on three key metabolic pathways in Estrecholia coli (E. coli), we are now able to undertake more safe and expedient direct evolution on organisms. The modular design and flexible functionality of our systems have empowered us to rapidly explore and optimize target genetic elements, unlocking new possibilities for directed evolution.
Our parts collection can be divided into two components, the Basic Parts and the Composite Parts.
The Basic Parts mainly involve the fusion proteins of our first-generation black box system and second-generation black box system, and the key enzymes in three metabolic pathways.
The Composite Parts contains three parts: the first-generation black box system, the second-generation black box system, and the metabolic pathways.
The detailed document of our Basic Parts and Composite Parts are listed below.
| Parts Name | Number | Description |
|---|---|---|
| rAPOBEC1-RNAP fusion protein | BBa_K5191000 | rAPOBEC1-RNAP fusion protein is a biotechnological tool that combines a cytidine base editor (CBE) with RNA polymerase (RNAP). This fusion allows for the targeted editing of DNA sequences by enabling the conversion of specific cytidine residues to thymine in DNA. The rAPOBEC1 component utilizes a deaminase enzyme to facilitate this conversion, while the RNAP component is responsible for synthesizing the RNA. By integrating base editing capabilities with RNA synthesize, rAPOBEC1-N terminal RNAP-C split RNAP represents a powerful strategy for gene editing. |
| rAPOBEC1-nMagHigh1-N-terminal RNAP fusion protein | BBa_K5191001 | The rAPOBEC1-nMagHigh1-N-terminal RNAP fusion protein is a tool in gene editing that combines the cytidine deaminase rAPOBEC1 with nMagHigh1, a photoresponsive protein, and N-terminal RNA polymerase (RNAP). The nMagHigh1 component can combine with pMagHigh1 under blue light, providing a means to control the activity of the fusion protein spatially and temporally. This fusion allows for RNA synthesize with precise DNA editing by enabling rAPOBEC1 to convert cytidine (C) to thymine (T) in DNA under blue light. By utilizing light to activate or deactivate the protein, researchers can achieve targeted modifications in DNA, enhancing the specificity of gene editing. |
| pMagHigh1-C-split RNAP fusion protein | BBa_K5191002 | The pMagHigh1-C-terminal RNAP fusion protein is a tool in gene editing that combines pMagHigh1, a photoresponsive protein, with C-terminal RNA polymerase (RNAP). The pMagHigh1 component can combine with nMagHigh1 under blue light, providing a means to control the activity of the fusion protein spatially and temporally. This fusion allows for controllable RNA synthesize by utilizing blue light to activate or deactivate the protein. |
| spermidine synthase | BBa_K5191004 | Spermidine synthase is a key enzyme involved in polyamine biosynthesis, specifically converting putrescine into spermidine. This enzyme catalyzes the reaction between putrescine and decarboxylated S-adenosylmethionine (dcSAM), resulting in the formation of spermidine and methylthioadenosine. Spermidine plays a crucial role in cell growth and proliferation by influencing cell cycle regulation, gene expression, and responses to oxidative stress and nutrient deprivation. Additionally, it is associated with autophagy processes, potentially affecting cellular aging and lifespan, and it exhibits neuroprotective effects on neural cells. These functions highlight the significant role of spermidine synthase in cellular physiology and medical research. |
| S-adenosylmethionine decarboxylase | BBa_K5191005 | S-adenosylmethionine decarboxylase (SAMDC) is a crucial enzyme in polyamine biosynthesis, catalyzing the decarboxylation of S-adenosylmethionine (SAM) to produce decarboxylated S-adenosylmethionine (dcSAM). This reaction is essential for the synthesis of polyamines like spermidine and spermine, which play vital roles in cellular functions such as growth, differentiation, and gene regulation. By facilitating polyamine production, SAMDC promotes cell proliferation and enhances cellular responses to stress, including oxidative damage. |
| agmatinase | BBa_K5191006 | Agmatinase is an enzyme that catalyzes the hydrolysis of agmatine to produce putrescine and urea. In the context of arginine metabolism, agmatinase plays a critical role in the conversion of arginine to agmatine, which is then further processed to form polyamines such as spermidine and spermine. This enzymatic pathway is essential for cellular growth, differentiation, and the regulation of gene expression. By facilitating the synthesis of polyamines, agmatinase contributes to various physiological processes, including cell proliferation, tissue repair, and response to stress. |
| 6-phosphogluconolactonase | BBa_K5191007 | 6-Phosphogluconolactonase is an enzyme that catalyzes the hydrolysis of 6-phosphogluconolactone into 6-phosphogluconate, a crucial step in the pentose phosphate pathway. This pathway is essential for cellular metabolism as it generates NADPH and ribose-5-phosphate. NADPH is vital for anabolic reactions, including fatty acid and nucleotide synthesis, while ribose-5-phosphate is a precursor for nucleotide biosynthesis. By facilitating these reactions, 6-phosphogluconolactonase plays a significant role in maintaining cellular redox balance and supporting biosynthetic processes. |
| transaldolase | BBa_K5191008 | Transaldolase is an enzyme that catalyzes the transfer of a three-carbon unit from sedoheptulose-7-phosphate to glyceraldehyde-3-phosphate, resulting in the formation of erythrose-4-phosphate and fructose-6-phosphate. This reaction is a key step in the pentose phosphate pathway and is crucial for the interconversion of sugars, facilitating the balance between nucleotide and amino acid synthesis. By providing essential intermediates for various biosynthetic pathways, transaldolase plays a significant role in cellular metabolism and the regulation of metabolic flux. |
| transketolase | BBa_K5191009 | Transketolase is an essential enzyme in the pentose phosphate pathway that catalyzes the transfer of a two-carbon unit from a ketose sugar, such as xylulose-5-phosphate, to an aldose sugar, such as ribose-5-phosphate. This reaction results in the formation of glyceraldehyde-3-phosphate and sedoheptulose-7-phosphate. Transketolase plays a crucial role in carbohydrate metabolism by facilitating the interconversion of sugars, which is vital for the synthesis of nucleotides and amino acids. By generating key intermediates, transketolase contributes to the overall cellular metabolic balance and supports various biosynthetic pathways. |
| nicotinate dehydrogenase | BBa_K5191010 | Nicotinate dehydrogenase is an enzyme that catalyzes the oxidation of nicotinate (niacin) to form nicotinamide in the presence of electron acceptors. This reaction is a key step in the metabolism of nicotine and other pyridine derivatives. Nicotinate dehydrogenase plays a crucial role in the oxidative degradation of nicotine, facilitating its conversion into less toxic metabolites. By participating in the energy generation process through the electron transport chain, this enzyme contributes to cellular respiration and overall metabolic balance. |
| 6-hydroxynicotinate reductase | BBa_K5191011 | 6-Hydroxynicotinate reductase is an enzyme that catalyzes the reduction of 6-hydroxynicotinate to 6-hydroxynicotinamide, using NADH or NADPH as an electron donor. This reaction is significant in the metabolic pathway of nicotine and its derivatives, facilitating the detoxification and conversion of nicotine into less harmful compounds. By participating in this reduction process, 6-hydroxynicotinate reductase plays a crucial role in the overall metabolism of nicotine, contributing to the regulation of its levels in the body and minimizing its potential toxic effects. |
| enamidase | BBa_K5191012 | Enamidase is an enzyme that catalyzes the hydrolysis of enamides to produce corresponding carboxylic acids and amines. In the context of nicotine metabolism, enamidase plays a crucial role in the breakdown of nicotine and its derivatives by converting them into more polar, water-soluble compounds. This enzymatic activity facilitates the detoxification process, allowing for easier excretion from the body. By participating in the metabolism of nicotine, enamidase contributes to the overall regulation of nicotine levels, helping to mitigate its potential harmful effects |
| Parts Name | Number | Description |
|---|---|---|
| T7 promoter-LacO-RBS-rAPOBEC1-N-terminal RNAP-C-split RNAP fusion protein-8xHis-T7 terminator-Amp-R promoter-Amp-R-Amp-R-terminator | BBa_K5191013 | The T7 promoter-LacO-RBS-rAPOBEC1-N-terminal RNAP-C-split RNAP-Ampicillin construct is a genetic system designed for regulated gene expression and targeted DNA editing. The T7 promoter initiates transcription in the presence of T7 RNA polymerase, enabling the gene editing in specific area only.The LacO operator allows for controlled expression in response to specific inducers, such as IPTG. The RBS (ribosome binding site) enhances translation efficiency, ensuring adequate production of the rAPOBEC1 enzyme, a cytidine deaminase that converts cytidine (C) to thymine (T) in DNA. The N-terminal RNAP and C-split RNAP components allows RNA synthesis. The 8xHis tag is a polyhistidine sequence used as a purification and detection tag. The T7 terminator acts as a sequence that signals the end of transcription for T7 RNA polymerase. The inclusion of Ampicillin resistance provides a selection marker, enabling the identification of successfully transformed cells. |
| T7 promoter-LacO-RBS-rAPOBEC1-nMagHigh1-N-terminal RNAP fusion protein-T7 promoter-LacO-RBS-pMagHigh1-C-split RNAP fusion protein-T7 promoter-LacO-EGFP-T7 terminator-Amp-R promoter-Amp-R-Amp-R-terminator | BBa_K5191015 | The construct comprising T7 promoter-LacO-RBS-rAPOBEC1-nMagHigh1-N-terminal RNAP fusion protein-T7 promoter-LacO-RBS-pMagHigh1-C-split RNAP fusion protein-T7 promoter-LacO-EGFP-Ampicillin is a comprehensive genetic system designed for advanced gene editing. The T7 promoter initiates transcription, enabling the gene editing in specific areas only. The LacO operator allows for tightly controlled expression of the downstream genes in response to inducers like IPTG. The RBS (ribosome binding site) enhances translation efficiency for the rAPOBEC1, nMagHigh1 and pMagHigh1, where rAPOBEC1 facilitates targeted DNA editing by converting cytidine (C) to thymine (T). nMagHigh1 and pMagHigh1 can combine together under blue light, along with N-terminal RNAP and C-split RNAP that synthesize proteins. The split RNAP system combines the two functional halves of RNA polymerase, enhancing transcription regulation. The construct also includes EGFP, a fluorescent marker that allows for real-time visualization of gene expression, and the Ampicillin resistance gene serves as a selection marker to identify successfully transformed cells. The T7 terminator acts as a sequence that signals the end of transcription for T7 RNA polymerase. |
| T7 promoter-RBS-spermidine synthase-RBS-S-adenosylmethionine decarboxylase-RBS-agmatinase-KEEP-T7 terminator；T7 promoter-RBS-6-phosphogluconolactonase -RBS-transaldolase-RBS-transketolase-KEEP-T7 terminator；T7 promoter-RBS-nicotinate dehydrogenase-RBS-6-hydroxynicotinate reductase-RBS-enamidase-KEEP-T7 terminator | BBa_K5191014 | The T7 promoter-RBS-spermidine synthase-RBS-S-adenosylmethionine decarboxylase-RBS-agmatinase-KEEP-T7 terminator construct is designed for the efficient production of polyamines, which are essential for various cellular processes. The T7 promoter initiates transcription in the presence of T7 RNA polymerase, while the ribosome binding sites (RBS) enhance the translation efficiency of the downstream enzymes: spermidine synthase, S-adenosylmethionine decarboxylase, and agmatinase. The S-adenosylmethionine decarboxylase converts S-adenosylmethionine (SAM) into decarboxylated SAM, which is a critical substrate for spermidine synthase, enabling it to catalyze the formation of spermidine from putrescine and decarboxylated SAM. Agmatinase then catalyzes the hydrolysis of agmatine into putrescine, completing the polyamine biosynthesis pathway. Additionally, the construct includes a KEEP (Kinetically Engineered Enzyme for Polyamine Production) element that optimizes the activity and stability of the enzymes involved, enhancing the overall efficiency of polyamine production. The T7 terminator ensures the proper termination of transcription, preventing read-through into adjacent genes. The T7 promoter-RBS-6-phosphogluconolactonase-RBS-transaldolase-RBS-transketolase-KEEP-T7 terminator construct is designed for efficient carbon flux through the pentose phosphate pathway (PPP), which is critical for the biosynthesis of nucleotides and amino acids. The T7 promoter initiates transcription in the presence of T7 RNA polymerase, while the ribosome binding sites (RBS) enhance the translation efficiency of the downstream enzymes: 6-phosphogluconolactonase, which catalyzes the hydrolysis of 6-phosphogluconolactone to produce 6-phosphogluconate, facilitating the conversion of glucose-6-phosphate into ribulose-5-phosphate; transaldolase, which transfers a three-carbon unit from sedoheptulose-7-phosphate to glyceraldehyde-3-phosphate, generating erythrose-4-phosphate and fructose-6-phosphate; and transketolase, which catalyzes the transfer of a two-carbon unit from xylulose-5-phosphate to ribose-5-phosphate, further contributing to the generation of intermediates for nucleotide and amino acid synthesis. The KEEP element enhances the activity and stability of these enzymes, improving overall metabolic efficiency. The T7 terminator ensures proper termination of transcription, preventing read-through into adjacent genes. The T7 promoter-RBS-nicotinate dehydrogenase-RBS-6-hydroxynicotinate reductase-RBS-enamidase-KEEP-T7 terminator construct is engineered for efficient conversion of nicotinic acid into valuable biochemical products, facilitating studies in metabolic pathways and synthetic biology. The T7 promoter initiates transcription in the presence of T7 RNA polymerase, while the ribosome binding sites (RBS) enhance the translation efficiency of the downstream enzymes: nicotinate dehydrogenase, which catalyzes the oxidation of nicotinic acid to 6-hydroxynicotinic acid; 6-hydroxynicotinate reductase, which reduces 6-hydroxynicotinic acid to 6-amino-2-pyridinecarboxylic acid; and enamidase, which hydrolyzes enamides to yield corresponding carboxylic acids. The KEEP element further optimizes the activity and stability of these enzymes, enhancing overall metabolic efficiency. The T7 terminator ensures proper termination of transcription, preventing read-through into adjacent genes. |
We implement the Second generation system on three practical matters for substantial evidence of our system; Using the second-generation black box, we targeted the pentose phosphate and polyamine pathways to boost NADPH and spermidine production. Sequencing confirmed mutations, and lifespan assays on Caenorhabditis elegans (C. elegans) showed extended lifespans due to increased metabolite production. For example, induced mutations in spermidine metabolic pathway such as A66T in spermidine synthase and R244C in 6-phosphogluconolactonase likely enhance metabolic efficiency, increasing spermidine production through synergistic effects. Similarly, a bio-brick containing enzymes for nicotinate degradation was mutated using the second-generation black box. Sequencing revealed C-to-T mutations in key enzymes, and lifespan assays confirmed mutant E. coli protected C. elegans from nicotinate toxicity. For specific results see here