Difference between revisions of "Part:BBa K4011019"
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<partinfo>BBa_K4011019 short</partinfo> | <partinfo>BBa_K4011019 short</partinfo> | ||
| − | pTALEsp2-TnaA-FL-FMO is an expression cassette in <i>E. coli</i> expressing TnaA-FL-FMO <partinfo>BBa_K4011004</partinfo> used to convert 6-Halogen-Trpytophan (6-X-Trp) into di-halogenated indigoid dyes such as tyrian purple. pTALEsp2 <partinfo> | + | pTALEsp2-TnaA-FL-FMO is an expression cassette in <i>E. coli</i> expressing TnaA-FL-FMO <partinfo>BBa_K4011004</partinfo> used to convert 6-Halogen-Trpytophan (6-X-Trp) into di-halogenated indigoid dyes such as tyrian purple. pTALEsp2 <partinfo>BBa_K2753019</partinfo> is an constitutive promoter suited to all strains of <i>E. coli</i>. B0015 <partinfo>BBa_B0015</partinfo> is a strong terminator. This is a part in a part collection where we enable the production of indigo, tyrian purple, and related dyes from tryptophan in <i>E. coli</i>. |
The part collection includes: Parts expressing Fre-SttH to convert Trp to 6-X-Trp. <partinfo>BBa_K4011003</partinfo> and <partinfo>BBa_K4011012</partinfo>. Parts expressing fusion protein TnaA-FMO to convert 6-X-Trp into indigoid dyes. <partinfo>BBa_K4011004</partinfo>, <partinfo>BBa_K4011005</partinfo>, <partinfo>BBa_K4011013</partinfo>, <partinfo>BBa_K4011014</partinfo>, <partinfo>BBa_K4011015</partinfo>, and <partinfo>BBa_K4011019</partinfo>. | The part collection includes: Parts expressing Fre-SttH to convert Trp to 6-X-Trp. <partinfo>BBa_K4011003</partinfo> and <partinfo>BBa_K4011012</partinfo>. Parts expressing fusion protein TnaA-FMO to convert 6-X-Trp into indigoid dyes. <partinfo>BBa_K4011004</partinfo>, <partinfo>BBa_K4011005</partinfo>, <partinfo>BBa_K4011013</partinfo>, <partinfo>BBa_K4011014</partinfo>, <partinfo>BBa_K4011015</partinfo>, and <partinfo>BBa_K4011019</partinfo>. | ||
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Our part collection can be used to help and inspire future teams to design and perfect different indigoid dye production pathways in <i>E. coli</i>, adding to the collection. | Our part collection can be used to help and inspire future teams to design and perfect different indigoid dye production pathways in <i>E. coli</i>, adding to the collection. | ||
| + | ==Usage and Biology== | ||
| − | < | + | TnaA is an tryptophanase found in <i>E. coli</i>. It converts tryptophan (trp) and other related molecules, such as 6-Halogen-trp (6-X-trp) into indole or 6-X-indole. Its specific reaction formula is L-tryptophan + H₂O ⇌ indole + pyruvate + NH₃. Indole, play important roles in signaling in bacterial cells. FMO is a monooxygenase found in <i>Methylophaga aminisulfidivorans</i>. It adds a hydroxyl group onto numerous molecules, in our case adding a hydroxyl onto the third carbon on indole or 6-X-indole, allowing for spontaneous dimerization of 3-hydroxyl-indole or 3-hydroxyl-6-X-indole into indigo or tyrian purple dyes. TnaA and FMO are fused together with the common flexible linker GGGGSGGGGS(Lee et al, 2021). |
| − | === | + | |
| + | The pTALEsp2 promoter is a constitutive promoter which self stabilizes, negating the effect of copy number on gene expression, thus maintaining gene expression at a stable level. For more information, visit <partinfo>BBa_K2753019</partinfo>. | ||
| + | |||
| + | ===Source=== | ||
| + | |||
| + | Tryptophanase (TnaA) is from <i>E. coli</i> and flavin-containing monooxygenase (FMO) is from <i>Methylophaga aminisulfidivorans</i>. | ||
| + | |||
| + | ==Design Considerations== | ||
| + | |||
| + | 1. All codons were optimized for <i>E. coli</i> based on <i>E. coli</i> codon bias. | ||
| + | |||
| + | 2. The sequences for the flexible linker is GGGGSGGGGS. | ||
| + | |||
| + | 3. Transformed and expressed in <i>E. coli DH5α ΔTnaA</i> to negate influence of endogenous TnaA in measurements. | ||
| + | |||
| + | ==Characterization== | ||
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Revision as of 12:15, 21 October 2021
pTALEsp2-TnaA-FL-FMO-B0015
pTALEsp2-TnaA-FL-FMO is an expression cassette in E. coli expressing TnaA-FL-FMO BBa_K4011004 used to convert 6-Halogen-Trpytophan (6-X-Trp) into di-halogenated indigoid dyes such as tyrian purple. pTALEsp2 BBa_K2753019 is an constitutive promoter suited to all strains of E. coli. B0015 BBa_B0015 is a strong terminator. This is a part in a part collection where we enable the production of indigo, tyrian purple, and related dyes from tryptophan in E. coli.
The part collection includes: Parts expressing Fre-SttH to convert Trp to 6-X-Trp. BBa_K4011003 and BBa_K4011012. Parts expressing fusion protein TnaA-FMO to convert 6-X-Trp into indigoid dyes. BBa_K4011004, BBa_K4011005, BBa_K4011013, BBa_K4011014, BBa_K4011015, and BBa_K4011019.
Our part collection can be used to help and inspire future teams to design and perfect different indigoid dye production pathways in E. coli, adding to the collection.
Usage and Biology
TnaA is an tryptophanase found in E. coli. It converts tryptophan (trp) and other related molecules, such as 6-Halogen-trp (6-X-trp) into indole or 6-X-indole. Its specific reaction formula is L-tryptophan + H₂O ⇌ indole + pyruvate + NH₃. Indole, play important roles in signaling in bacterial cells. FMO is a monooxygenase found in Methylophaga aminisulfidivorans. It adds a hydroxyl group onto numerous molecules, in our case adding a hydroxyl onto the third carbon on indole or 6-X-indole, allowing for spontaneous dimerization of 3-hydroxyl-indole or 3-hydroxyl-6-X-indole into indigo or tyrian purple dyes. TnaA and FMO are fused together with the common flexible linker GGGGSGGGGS(Lee et al, 2021).
The pTALEsp2 promoter is a constitutive promoter which self stabilizes, negating the effect of copy number on gene expression, thus maintaining gene expression at a stable level. For more information, visit BBa_K2753019.
Source
Tryptophanase (TnaA) is from E. coli and flavin-containing monooxygenase (FMO) is from Methylophaga aminisulfidivorans.
Design Considerations
1. All codons were optimized for E. coli based on E. coli codon bias.
2. The sequences for the flexible linker is GGGGSGGGGS.
3. Transformed and expressed in E. coli DH5α ΔTnaA to negate influence of endogenous TnaA in measurements.
Characterization
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 1513
Illegal NheI site found at 1921 - 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 113
Illegal BamHI site found at 5978
Illegal XhoI site found at 2152
Illegal XhoI site found at 2692 - 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal AgeI site found at 747
Illegal AgeI site found at 849
Illegal AgeI site found at 1767
Illegal AgeI site found at 2277
Illegal AgeI site found at 4385 - 1000COMPATIBLE WITH RFC[1000]