Parts

MsbA:

1.ATP-binding cassette (ABC) transporters MsbA （self-expression system with T7 promoter）[BBa_K1402002]

Function

ATP-binding cassette (ABC) transporters are integral membrane proteins that translocate a wide variety of substrates across cellular membranes and are conserved from bacteria to humans. This ABC transporters MsbA can transport fatty acids out of the cell.

Usage and Biology

The RBS and T7 promoter are in front of the MsbA coding sequence and T7 terminator is after MsbA. You can only use this part in E.coil BL21(DE3) or choose other parts we offer in other strains.

Sequence and Features

Tctagag [prefix]

GCGGCCGCTTCTAGATAATACGACTCACTATAGG [T7 promoter]

AAAGAGGAGAAA [RBS]

ATGCATAACGACAAAGATCTCTCTACGTGGCAGACATTCCGCCGACTGTGGCCAACCATTGCGCCTTTCAAAGCGGGTCT GATCGTGGCGGGCGTAGCGTTAATCCTCAACGCAGCCAGCGATACCTTCATGTTATCGCTCCTTAAGCCACTTCTTGATG ATGGCTTTGGTAAAACAGATCGCTCCGTGCTGGTGTGGATGCCGCTGGTGGTGATCGGGCTGATGATTTTACGTGGTATC ACCAGCTATGTCTCCAGCTACTGTATCTCCTGGGTATCAGGAAAGGTGGTAATGACCATGCGTCGCCGCCTGTTTGGTCA CATGATGGGAATGCCAGTTTCATTCTTTGACAAACAGTCAACGGGTACGCTGTTGTCACGTATTACCTACGATTCCGAAC AGGTTGCTTCTTCTTCTTCCGGCGCACTGATTACTGTTGTGCGTGAAGGTGCGTCGATCATCGGCCTGTTCATCATGATG TTCTATTACAGTTGGCAACTGTCGATCATTTTGATTGTGCTGGCACCGATTGTTTCGATTGCGATTCGCGTTGTATCGAA GCGTTTTCGCAACATCAGTAAAAACATGCAGAACACCATGGGGCAGGTGACCACCAGCGCAGAACAAATGCTGAAGGGCC ACAAAGAAGTATTGATTTTCGGTGGTCAGGAAGTGGAAACGAAACGCTTTGATAAAGTCAGCAACCGAATGCGTCTTCAG GGGATGAAAATGGTTTCAGCCTCTTCCATCTCTGATCCGATCATTCAGCTGATCGCCTCTTTGGCGCTGGCGTTTGTTCT GTATGCGGCGAGCTTCCCAAGTGTCATGGATAGCCTGACTGCCGGTACGATTACCGTTGTTTTCTCTTCAATGATTGCAC TGATGCGTCCGCTGAAATCGCTGACCAACGTTAACGCCCAGTTCCAGCGCGGTATGGCGGCTTGTCAGACGCTGTTTACC ATTCTGGACAGTGAGCAGGAGAAAGATGAAGGTAAGCGCGTGATCGAGCGTGCGACTGGCGACGTGGAGTTCCGCAATGT CACCTTTACTTATCCGGGACGTGACGTACCTGCATTGCGTAACATCAACCTGAAAATTCCGGCAGGGAAGACGGTTGCTC TGGTTGGACGCTCTGGTTCGGGTAAATCAACCATCGCCAGCCTGATCACGCGTTTTTACGATATTGATGAAGGCGAAATC CTGATGGATGGTCACGATCTGCGCGAGTATACCCTGGCGTCGTTACGTAACCAGGTTGCTCTGGTGTCGCAGAATGTCCA TCTGTTTAACGATACGGTTGCTAACAACATTGCTTACGCACGGACTGAACAGTACAGCCGTGAGCAAATTGAAGAAGCGG CGCGTATGGCCTACGCCATGGACTTCATCAATAAGATGGATAACGGTCTCGATACAGTGATTGGTGAAAACGGCGTGCTG CTCTCTGGCGGTCAGCGTCAGCGTATTGCTATCGCTCGAGCCTTGTTGCGTGATAGCCCGATTCTGATTCTGGACGAAGC TACCTCGGCTCTGGATACCGAATCCGAACGTGCGATTCAGGCGGCACTGGATGAGTTGCAGAAAAACCGTACCTCTCTGG TGATTGCCCACCGCTTGTCTACCATTGAAAAGGCAGACGAAATCGTGGTCGTCGAGGATGGTGTCATTGTGGAACGCGGT ACGCATAACGATTTGCTTGAGCACCGCGGCGTTTACGCGCAACTTCACAAAATGCAGTTTGGCCAATGA [MsbA CDS]

CTGCTAACAAAGCCCGAAAGGAAGCTGAGTTGGCTGCTGCCACCGCTGAGCAATAACTAGCATAACCCCTTGGGGCCTCT AAACGGGTCTTGAGGGGTTTTTTGCTGAAAGGAGGAACTATATCCGGAT[T7 terminator]

Tactagt [suffix]

2.ATP-binding cassette (ABC) transporters MsbA （with a promoter absent）

Function

ATP-binding cassette (ABC) transporters are integral membrane proteins that translocate a wide variety of substrates across cellular membranes and are conserved from bacteria to humans.this ABC transporters MsbA can transport fatty acids out of the cell.

Usage and Biology

The RBS is in front of the MsbA coding sequence and T7 terminator is after MsbA. You can design your own promoter for this part ,but use this part in E.coil BL21(DE3) only or you’d better delete the T7 terminator.

Sequence and Features

Tctagag [prefix]

AAAGAGGAGAAA [RBS]

ATGCATAACGACAAAGATCTCTCTACGTGGCAGACATTCCGCCGACTGTGGCCAACCATTGCGCCTTTCAAAGCGGGTCT GATCGTGGCGGGCGTAGCGTTAATCCTCAACGCAGCCAGCGATACCTTCATGTTATCGCTCCTTAAGCCACTTCTTGATG ATGGCTTTGGTAAAACAGATCGCTCCGTGCTGGTGTGGATGCCGCTGGTGGTGATCGGGCTGATGATTTTACGTGGTATC ACCAGCTATGTCTCCAGCTACTGTATCTCCTGGGTATCAGGAAAGGTGGTAATGACCATGCGTCGCCGCCTGTTTGGTCA CATGATGGGAATGCCAGTTTCATTCTTTGACAAACAGTCAACGGGTACGCTGTTGTCACGTATTACCTACGATTCCGAAC AGGTTGCTTCTTCTTCTTCCGGCGCACTGATTACTGTTGTGCGTGAAGGTGCGTCGATCATCGGCCTGTTCATCATGATG TTCTATTACAGTTGGCAACTGTCGATCATTTTGATTGTGCTGGCACCGATTGTTTCGATTGCGATTCGCGTTGTATCGAA GCGTTTTCGCAACATCAGTAAAAACATGCAGAACACCATGGGGCAGGTGACCACCAGCGCAGAACAAATGCTGAAGGGCC ACAAAGAAGTATTGATTTTCGGTGGTCAGGAAGTGGAAACGAAACGCTTTGATAAAGTCAGCAACCGAATGCGTCTTCAG GGGATGAAAATGGTTTCAGCCTCTTCCATCTCTGATCCGATCATTCAGCTGATCGCCTCTTTGGCGCTGGCGTTTGTTCT GTATGCGGCGAGCTTCCCAAGTGTCATGGATAGCCTGACTGCCGGTACGATTACCGTTGTTTTCTCTTCAATGATTGCAC TGATGCGTCCGCTGAAATCGCTGACCAACGTTAACGCCCAGTTCCAGCGCGGTATGGCGGCTTGTCAGACGCTGTTTACC ATTCTGGACAGTGAGCAGGAGAAAGATGAAGGTAAGCGCGTGATCGAGCGTGCGACTGGCGACGTGGAGTTCCGCAATGT CACCTTTACTTATCCGGGACGTGACGTACCTGCATTGCGTAACATCAACCTGAAAATTCCGGCAGGGAAGACGGTTGCTC TGGTTGGACGCTCTGGTTCGGGTAAATCAACCATCGCCAGCCTGATCACGCGTTTTTACGATATTGATGAAGGCGAAATC CTGATGGATGGTCACGATCTGCGCGAGTATACCCTGGCGTCGTTACGTAACCAGGTTGCTCTGGTGTCGCAGAATGTCCA TCTGTTTAACGATACGGTTGCTAACAACATTGCTTACGCACGGACTGAACAGTACAGCCGTGAGCAAATTGAAGAAGCGG CGCGTATGGCCTACGCCATGGACTTCATCAATAAGATGGATAACGGTCTCGATACAGTGATTGGTGAAAACGGCGTGCTG CTCTCTGGCGGTCAGCGTCAGCGTATTGCTATCGCTCGAGCCTTGTTGCGTGATAGCCCGATTCTGATTCTGGACGAAGC TACCTCGGCTCTGGATACCGAATCCGAACGTGCGATTCAGGCGGCACTGGATGAGTTGCAGAAAAACCGTACCTCTCTGG TGATTGCCCACCGCTTGTCTACCATTGAAAAGGCAGACGAAATCGTGGTCGTCGAGGATGGTGTCATTGTGGAACGCGGT ACGCATAACGATTTGCTTGAGCACCGCGGCGTTTACGCGCAACTTCACAAAATGCAGTTTGGCCAATGA [MsbA CDS]

CTGCTAACAAAGCCCGAAAGGAAGCTGAGTTGGCTGCTGCCACCGCTGAGCAATAACTAGCATAACCCCTTGGGGCCTCT AAACGGGTCTTGAGGGGTTTTTTGCTGAAAGGAGGAACTATATCCGGAT [T7 terminator]

Tactagt [suffix]

3.ATP-binding cassette (ABC) transporters MsbA

Function

ATP-binding cassette (ABC) transporters are integral membrane proteins that translocate a wide variety of substrates across cellular membranes and are conserved from bacteria to humans.this ABC transporters MsbA can transport fatty acids out of the cell.

Usage and Biology

This is the coding squence of MsbA. It can not express itself. You should add promoter, RBS and terminator to it or use other parts we offer.

Sequence and Features

Tctagag [prefix]

ATGCATAACGACAAAGATCTCTCTACGTGGCAGACATTCCGCCGACTGTGGCCAACCATTGCGCCTTTCAAAGCGGGTCT GATCGTGGCGGGCGTAGCGTTAATCCTCAACGCAGCCAGCGATACCTTCATGTTATCGCTCCTTAAGCCACTTCTTGATG ATGGCTTTGGTAAAACAGATCGCTCCGTGCTGGTGTGGATGCCGCTGGTGGTGATCGGGCTGATGATTTTACGTGGTATC ACCAGCTATGTCTCCAGCTACTGTATCTCCTGGGTATCAGGAAAGGTGGTAATGACCATGCGTCGCCGCCTGTTTGGTCA CATGATGGGAATGCCAGTTTCATTCTTTGACAAACAGTCAACGGGTACGCTGTTGTCACGTATTACCTACGATTCCGAAC AGGTTGCTTCTTCTTCTTCCGGCGCACTGATTACTGTTGTGCGTGAAGGTGCGTCGATCATCGGCCTGTTCATCATGATG TTCTATTACAGTTGGCAACTGTCGATCATTTTGATTGTGCTGGCACCGATTGTTTCGATTGCGATTCGCGTTGTATCGAA GCGTTTTCGCAACATCAGTAAAAACATGCAGAACACCATGGGGCAGGTGACCACCAGCGCAGAACAAATGCTGAAGGGCC ACAAAGAAGTATTGATTTTCGGTGGTCAGGAAGTGGAAACGAAACGCTTTGATAAAGTCAGCAACCGAATGCGTCTTCAG GGGATGAAAATGGTTTCAGCCTCTTCCATCTCTGATCCGATCATTCAGCTGATCGCCTCTTTGGCGCTGGCGTTTGTTCT GTATGCGGCGAGCTTCCCAAGTGTCATGGATAGCCTGACTGCCGGTACGATTACCGTTGTTTTCTCTTCAATGATTGCAC TGATGCGTCCGCTGAAATCGCTGACCAACGTTAACGCCCAGTTCCAGCGCGGTATGGCGGCTTGTCAGACGCTGTTTACC ATTCTGGACAGTGAGCAGGAGAAAGATGAAGGTAAGCGCGTGATCGAGCGTGCGACTGGCGACGTGGAGTTCCGCAATGT CACCTTTACTTATCCGGGACGTGACGTACCTGCATTGCGTAACATCAACCTGAAAATTCCGGCAGGGAAGACGGTTGCTC TGGTTGGACGCTCTGGTTCGGGTAAATCAACCATCGCCAGCCTGATCACGCGTTTTTACGATATTGATGAAGGCGAAATC CTGATGGATGGTCACGATCTGCGCGAGTATACCCTGGCGTCGTTACGTAACCAGGTTGCTCTGGTGTCGCAGAATGTCCA TCTGTTTAACGATACGGTTGCTAACAACATTGCTTACGCACGGACTGAACAGTACAGCCGTGAGCAAATTGAAGAAGCGG CGCGTATGGCCTACGCCATGGACTTCATCAATAAGATGGATAACGGTCTCGATACAGTGATTGGTGAAAACGGCGTGCTG CTCTCTGGCGGTCAGCGTCAGCGTATTGCTATCGCTCGAGCCTTGTTGCGTGATAGCCCGATTCTGATTCTGGACGAAGC TACCTCGGCTCTGGATACCGAATCCGAACGTGCGATTCAGGCGGCACTGGATGAGTTGCAGAAAAACCGTACCTCTCTGG TGATTGCCCACCGCTTGTCTACCATTGAAAAGGCAGACGAAATCGTGGTCGTCGAGGATGGTGTCATTGTGGAACGCGGT ACGCATAACGATTTGCTTGAGCACCGCGGCGTTTACGCGCAACTTCACAAAATGCAGTTTGGCCAATGA [MsbA CDS]

Tactagt [suffix]

4.ATP-binding cassette (ABC) transporters MsbA （regulated-expression system with FadD promoter）

Function

ATP-binding cassette (ABC) transporters are integral membrane proteins that translocate a wide variety of substrates across cellular membranes and are conserved from bacteria to humans.this ABC transporters MsbA can transport fatty acids out of the cell.

Usage and Biology

The RBS and FadD promoter are in front of the MsbA coding sequence and T7 terminator is after MsbA. The FadD promoter is sensitive to the concentration of fatty acid. Due to the over expression of MsbA can be harm to the growth of E.coli, it is activated only when the fatty acid concentration is high to lower the toxicity of MsbA.

Sequence and Features

Tctagag [prefix]

CGCTTCGCAACCTTTTCGTTGGGTAATTATCAAGCTGGTATGATGAGTTAATATTATGTT[FadD promoter]

AAAGAGGAGAAA [RBS]

ATGCATAACGACAAAGATCTCTCTACGTGGCAGACATTCCGCCGACTGTGGCCAACCATTGCGCCTTTCAAAGCGGGTCT GATCGTGGCGGGCGTAGCGTTAATCCTCAACGCAGCCAGCGATACCTTCATGTTATCGCTCCTTAAGCCACTTCTTGATG ATGGCTTTGGTAAAACAGATCGCTCCGTGCTGGTGTGGATGCCGCTGGTGGTGATCGGGCTGATGATTTTACGTGGTATC ACCAGCTATGTCTCCAGCTACTGTATCTCCTGGGTATCAGGAAAGGTGGTAATGACCATGCGTCGCCGCCTGTTTGGTCA CATGATGGGAATGCCAGTTTCATTCTTTGACAAACAGTCAACGGGTACGCTGTTGTCACGTATTACCTACGATTCCGAAC AGGTTGCTTCTTCTTCTTCCGGCGCACTGATTACTGTTGTGCGTGAAGGTGCGTCGATCATCGGCCTGTTCATCATGATG TTCTATTACAGTTGGCAACTGTCGATCATTTTGATTGTGCTGGCACCGATTGTTTCGATTGCGATTCGCGTTGTATCGAA GCGTTTTCGCAACATCAGTAAAAACATGCAGAACACCATGGGGCAGGTGACCACCAGCGCAGAACAAATGCTGAAGGGCC ACAAAGAAGTATTGATTTTCGGTGGTCAGGAAGTGGAAACGAAACGCTTTGATAAAGTCAGCAACCGAATGCGTCTTCAG GGGATGAAAATGGTTTCAGCCTCTTCCATCTCTGATCCGATCATTCAGCTGATCGCCTCTTTGGCGCTGGCGTTTGTTCT GTATGCGGCGAGCTTCCCAAGTGTCATGGATAGCCTGACTGCCGGTACGATTACCGTTGTTTTCTCTTCAATGATTGCAC TGATGCGTCCGCTGAAATCGCTGACCAACGTTAACGCCCAGTTCCAGCGCGGTATGGCGGCTTGTCAGACGCTGTTTACC ATTCTGGACAGTGAGCAGGAGAAAGATGAAGGTAAGCGCGTGATCGAGCGTGCGACTGGCGACGTGGAGTTCCGCAATGT CACCTTTACTTATCCGGGACGTGACGTACCTGCATTGCGTAACATCAACCTGAAAATTCCGGCAGGGAAGACGGTTGCTC TGGTTGGACGCTCTGGTTCGGGTAAATCAACCATCGCCAGCCTGATCACGCGTTTTTACGATATTGATGAAGGCGAAATC CTGATGGATGGTCACGATCTGCGCGAGTATACCCTGGCGTCGTTACGTAACCAGGTTGCTCTGGTGTCGCAGAATGTCCA TCTGTTTAACGATACGGTTGCTAACAACATTGCTTACGCACGGACTGAACAGTACAGCCGTGAGCAAATTGAAGAAGCGG CGCGTATGGCCTACGCCATGGACTTCATCAATAAGATGGATAACGGTCTCGATACAGTGATTGGTGAAAACGGCGTGCTG CTCTCTGGCGGTCAGCGTCAGCGTATTGCTATCGCTCGAGCCTTGTTGCGTGATAGCCCGATTCTGATTCTGGACGAAGC TACCTCGGCTCTGGATACCGAATCCGAACGTGCGATTCAGGCGGCACTGGATGAGTTGCAGAAAAACCGTACCTCTCTGG TGATTGCCCACCGCTTGTCTACCATTGAAAAGGCAGACGAAATCGTGGTCGTCGAGGATGGTGTCATTGTGGAACGCGGT ACGCATAACGATTTGCTTGAGCACCGCGGCGTTTACGCGCAACTTCACAAAATGCAGTTTGGCCAATGA [MsbA CDS]

CTGCTAACAAAGCCCGAAAGGAAGCTGAGTTGGCTGCTGCCACCGCTGAGCAATAACTAGCATAACCCCTTGGGGCCTCT AAACGGGTCTTGAGGGGTTTTTTGCTGAAAGGAGGAACTATATCCGGAT[T7 terminator]

Tactagt [suffix]

BTE:

1.ACP Thioesterase BTE

Function

Acyl carrier protein (ACP) thioesterase catalyzes the hydrolysis of acyl-ACP into fatty acid and ACP. This reaction removes the coupling between fatty acid biosynthesis and phospholipid biosyntheis, which is a key to obtaining free fatty acids. This thioesterase BTE can produce fatty acids with 12~14 carbon chain length. In previous reserches, thioesterase gene BTE can be expressed in E.coil BL21(DE3) and enhance the synthesis of free fatty acids.

Usage and Biology

This part can increase the productivity of free fatty acids. However,this part can not express itself. You should add promoter, RBS and terminator to it or using other parts we offer.

Sequence and Features

Tctagag [prefix]

AGAGAGAGAGAGAGAGAGAGAGCTAAATTAAAAAAAAAACCCAGAAGTGGGAAATCTTCCCCATGAAATAACGGATCCTC TTGCTACTGCTACTACTACTACTACAAACTGTAGCCATTTATATAATTCTATATAATTTTCAACATGGCCACCACCTCTT TAGCTTCCGCTTTCTGCTCGATGAAAGCTGTAATGTTGGCTCGTGATGGCCGGGGCATGAAACCCAGGAGCAGTGATTTG CAGCTGAGGGCGGGAAATGCGCCAACCTCTTTGAAGATGATCAATGGGACCAAGTTCAGTTACACGGAGAGCTTGAAAAG GTTGCCTGACTGGAGCATGCTCTTTGCAGTGATCACAACCATCTTTTCGGCTGCTGAGAAGCAGTGGACCAATCTGGAGT GGAAGCCGAAGCCGAAGCTACCCCAGTTGCTTGATGACCATTTTGGACTGCATGGGTTAGTTTTCAGGCGCACCTTTGCC ATCAGATCTTATGAGGTGGGACCTGACCGCTCCACATCTATACTGGCTGTTATGAATCACATGCAGGAGGCTACACTTAA TCATGCGAAGAGTGTGGGAATCCTAGGAGATGGATTCGGGACGACGCTAGAGATGAGTAAGAGAGATCTGATGTGGGTTG TGAGACGCACGCATGTTGCTGTGGAACGGTACCCTACTTGGGGTGATACTGTAGAAGTAGAGTGCTGGATTGGTGCATCT GGAAATAATGGCATGCGACGTGATTTCCTTGTCCGGGACTGCAAAACAGGCGAAATTCTTACAAGATGTACCAGCCTTTC GGTGCTGATGAATACAAGGACAAGGAGGTTGTCCACAATCCCTGACGAAGTTAGAGGGGAGATAGGGCCTGCATTCATTG ATAATGTGGCTGTCAAGGACGATGAAATTAAGAAACTACAGAAGCTCAATGACAGCACTGCCGATTACATCCAAGGAGGT TTGACTCCTCGATGGAATGATTTGGATGTCAATCAGCATGTGAACAACCTCAAATACGTTGCCTGGGTTTTTGAGACCGT CCCAGACTCCATCTTTGAGAGTCATCATATTTCCAGCTTCACTCTTGAATACAGGAGAGAGTGCACGAGGGATAGCGTGC TGCGGTCCCTGACCACTGTCTCTGGTGGCTCGTCGGAGGCTGGGTTAGTGTGCGATCACTTGCTCCAGCTTGAAGGTGGG TCTGAGGTATTGAGGGCAAGAACAGAGTGGAGGCCTAAGCTTACCGATAGTTTCAGAGGGATTAGTGTGATACCCGCAGA ACCGAGGGTGTAACTAATGAAAGAAGCATCTGTTGAAGTTTCTCCCATGCTGTTCGTGAGGATACTTTTTAGAAGCTGTA GTTTGCATTGCTTGTGCAGAATCATGGTCTGTGGTTTTAGATGTATATAAAAAATAGTCCTGTAGTCATGAAACTTAATA TCAGAAAAATAACTCAATGGGTCAAGGTTATCGAAGTAGTCATTTAAGCTTTGAAATATGTTTTGTATTCCTCGGCTTAA TCTGTAAGCTCTTTCTCTTGCAATAAAGTTCGCCTTTCAAT [BTE CDS]

Tactagt [suffix]

2.ACP Thioesterase BTE(self-expression system with T7 promoter)

Function

Acyl carrier protein (ACP) thioesterase catalyzes the hydrolysis of acyl-ACP into fatty acid and ACP. This reaction removes the coupling between fatty acid biosynthesis and phospholipid biosyntheis, which is a key to obtaining free fatty acids. This thioesterase BTE can produce fatty acids with 12~14 carbon chain length. In previous reserches, thioesterase gene BTE can be expressed in E.coil BL21(DE3) and enhance the synthesis of free fatty acids.

This thioesterase can express itself with a T7 promoter.

Usage and Biology

The RBS and T7 promoter are in front of the BTE coding sequence and T7 terminator is after BTE. You can only use this part in E.coil BL21(DE3) or choose other parts we offer in other strains.

Sequence and Features

Tctagag [prefix]

GCGGCCGCTTCTAGATAATACGACTCACTATAGG [T7 promoter]

AAAGAGGAGAAA [RBS]

AGAGAGAGAGAGAGAGAGAGAGCTAAATTAAAAAAAAAACCCAGAAGTGGGAAATCTTCCCCATGAAATAACGGATCCTC TTGCTACTGCTACTACTACTACTACAAACTGTAGCCATTTATATAATTCTATATAATTTTCAACATGGCCACCACCTCTT TAGCTTCCGCTTTCTGCTCGATGAAAGCTGTAATGTTGGCTCGTGATGGCCGGGGCATGAAACCCAGGAGCAGTGATTTG CAGCTGAGGGCGGGAAATGCGCCAACCTCTTTGAAGATGATCAATGGGACCAAGTTCAGTTACACGGAGAGCTTGAAAAG GTTGCCTGACTGGAGCATGCTCTTTGCAGTGATCACAACCATCTTTTCGGCTGCTGAGAAGCAGTGGACCAATCTGGAGT GGAAGCCGAAGCCGAAGCTACCCCAGTTGCTTGATGACCATTTTGGACTGCATGGGTTAGTTTTCAGGCGCACCTTTGCC ATCAGATCTTATGAGGTGGGACCTGACCGCTCCACATCTATACTGGCTGTTATGAATCACATGCAGGAGGCTACACTTAA TCATGCGAAGAGTGTGGGAATCCTAGGAGATGGATTCGGGACGACGCTAGAGATGAGTAAGAGAGATCTGATGTGGGTTG TGAGACGCACGCATGTTGCTGTGGAACGGTACCCTACTTGGGGTGATACTGTAGAAGTAGAGTGCTGGATTGGTGCATCT GGAAATAATGGCATGCGACGTGATTTCCTTGTCCGGGACTGCAAAACAGGCGAAATTCTTACAAGATGTACCAGCCTTTC GGTGCTGATGAATACAAGGACAAGGAGGTTGTCCACAATCCCTGACGAAGTTAGAGGGGAGATAGGGCCTGCATTCATTG ATAATGTGGCTGTCAAGGACGATGAAATTAAGAAACTACAGAAGCTCAATGACAGCACTGCCGATTACATCCAAGGAGGT TTGACTCCTCGATGGAATGATTTGGATGTCAATCAGCATGTGAACAACCTCAAATACGTTGCCTGGGTTTTTGAGACCGT CCCAGACTCCATCTTTGAGAGTCATCATATTTCCAGCTTCACTCTTGAATACAGGAGAGAGTGCACGAGGGATAGCGTGC TGCGGTCCCTGACCACTGTCTCTGGTGGCTCGTCGGAGGCTGGGTTAGTGTGCGATCACTTGCTCCAGCTTGAAGGTGGG TCTGAGGTATTGAGGGCAAGAACAGAGTGGAGGCCTAAGCTTACCGATAGTTTCAGAGGGATTAGTGTGATACCCGCAGA ACCGAGGGTGTAACTAATGAAAGAAGCATCTGTTGAAGTTTCTCCCATGCTGTTCGTGAGGATACTTTTTAGAAGCTGTA GTTTGCATTGCTTGTGCAGAATCATGGTCTGTGGTTTTAGATGTATATAAAAAATAGTCCTGTAGTCATGAAACTTAATA TCAGAAAAATAACTCAATGGGTCAAGGTTATCGAAGTAGTCATTTAAGCTTTGAAATATGTTTTGTATTCCTCGGCTTAA TCTGTAAGCTCTTTCTCTTGCAATAAAGTTCGCCTTTCAAT [BTE CDS]

CTGCTAACAAAGCCCGAAAGGAAGCTGAGTTGGCTGCTGCCACCGCTGAGCAATAACTAGCATAACCCCTTGGGGCCTCT AAACGGGTCTTGAGGGGTTTTTTGCTGAAAGGAGGAACTATATCCGGAT [T7 terminator]

Tactagt [suffix]

3.ACP Thioesterase BTE(with a promoter absent)

Function

Acyl carrier protein (ACP) thioesterase catalyzes the hydrolysis of acyl-ACP into fatty acid and ACP. This reaction removes the coupling between fatty acid biosynthesis and phospholipid biosyntheis, which is a key to obtaining free fatty acids. This thioesterase BTE can produce fatty acids with 12~14 carbon chain length. In previous reserches, thioesterase gene BTE can be expressed in E.coil BL21(DE3) and enhance the synthesis of free fatty acids.

This thioesterase can not express itself.

Usage and Biology

The RBS is in front of the BTE coding sequence and T7 terminator is after BTE. You can design your own promoter for this part, but use this part in E.coil BL21(DE3) only or you’d better delete the T7 terminator.

Sequence and Features

Tctagag [prefix]

AAAGAGGAGAAA [RBS]

AGAGAGAGAGAGAGAGAGAGAGCTAAATTAAAAAAAAAACCCAGAAGTGGGAAATCTTCCCCATGAAATAACGGATCCTC TTGCTACTGCTACTACTACTACTACAAACTGTAGCCATTTATATAATTCTATATAATTTTCAACATGGCCACCACCTCTT TAGCTTCCGCTTTCTGCTCGATGAAAGCTGTAATGTTGGCTCGTGATGGCCGGGGCATGAAACCCAGGAGCAGTGATTTG CAGCTGAGGGCGGGAAATGCGCCAACCTCTTTGAAGATGATCAATGGGACCAAGTTCAGTTACACGGAGAGCTTGAAAAG GTTGCCTGACTGGAGCATGCTCTTTGCAGTGATCACAACCATCTTTTCGGCTGCTGAGAAGCAGTGGACCAATCTGGAGT GGAAGCCGAAGCCGAAGCTACCCCAGTTGCTTGATGACCATTTTGGACTGCATGGGTTAGTTTTCAGGCGCACCTTTGCC ATCAGATCTTATGAGGTGGGACCTGACCGCTCCACATCTATACTGGCTGTTATGAATCACATGCAGGAGGCTACACTTAA TCATGCGAAGAGTGTGGGAATCCTAGGAGATGGATTCGGGACGACGCTAGAGATGAGTAAGAGAGATCTGATGTGGGTTG TGAGACGCACGCATGTTGCTGTGGAACGGTACCCTACTTGGGGTGATACTGTAGAAGTAGAGTGCTGGATTGGTGCATCT GGAAATAATGGCATGCGACGTGATTTCCTTGTCCGGGACTGCAAAACAGGCGAAATTCTTACAAGATGTACCAGCCTTTC GGTGCTGATGAATACAAGGACAAGGAGGTTGTCCACAATCCCTGACGAAGTTAGAGGGGAGATAGGGCCTGCATTCATTG ATAATGTGGCTGTCAAGGACGATGAAATTAAGAAACTACAGAAGCTCAATGACAGCACTGCCGATTACATCCAAGGAGGT TTGACTCCTCGATGGAATGATTTGGATGTCAATCAGCATGTGAACAACCTCAAATACGTTGCCTGGGTTTTTGAGACCGT CCCAGACTCCATCTTTGAGAGTCATCATATTTCCAGCTTCACTCTTGAATACAGGAGAGAGTGCACGAGGGATAGCGTGC TGCGGTCCCTGACCACTGTCTCTGGTGGCTCGTCGGAGGCTGGGTTAGTGTGCGATCACTTGCTCCAGCTTGAAGGTGGG TCTGAGGTATTGAGGGCAAGAACAGAGTGGAGGCCTAAGCTTACCGATAGTTTCAGAGGGATTAGTGTGATACCCGCAGA ACCGAGGGTGTAACTAATGAAAGAAGCATCTGTTGAAGTTTCTCCCATGCTGTTCGTGAGGATACTTTTTAGAAGCTGTA GTTTGCATTGCTTGTGCAGAATCATGGTCTGTGGTTTTAGATGTATATAAAAAATAGTCCTGTAGTCATGAAACTTAATA TCAGAAAAATAACTCAATGGGTCAAGGTTATCGAAGTAGTCATTTAAGCTTTGAAATATGTTTTGTATTCCTCGGCTTAA TCTGTAAGCTCTTTCTCTTGCAATAAAGTTCGCCTTTCAAT [BTE CDS]

CTGCTAACAAAGCCCGAAAGGAAGCTGAGTTGGCTGCTGCCACCGCTGAGCAATAACTAGCATAACCCCTTGGGGCCTCT AAACGGGTCTTGAGGGGTTTTTTGCTGAAAGGAGGAACTATATCCGGAT[T7 terminator]

Tactagt [suffix]

4.ACP Thioesterase BTE(regulated-expression system with ara promoter)

Function

Acyl carrier protein (ACP) thioesterase catalyzes the hydrolysis of acyl-ACP into fatty acid and ACP. This reaction removes the coupling between fatty acid biosynthesis and phospholipid biosyntheis, which is a key to obtaining free fatty acids. This thioesterase BTE can produce fatty acids with 12~14 carbon chain length. In previous reserches, thioesterase gene BTE can be expressed in E.coil BL21(DE3) and enhance the synthesis of free fatty acids.

Usage and Biology

The RBS and ara promoter are before the BTE coding sequence and T7 terminator is after BTE. We can activate the expression of BTE by adding arabinose in the culture in the absence of glucose.

Sequence and Features

Tctagag [prefix]

AAACCAATTGTCCATATTGCATCAGACATTGCCGTCACTGCGTCTTTTACTGGCTCTTCTCGCTAACCAAACCGGTAACC CCGCTTATTAAAAGCATTCTGTAACAAAGCGGGACCAAAGCCATGACAAAAACGCGTAACAAAAGTGTCTATAATCACGG CAGAAAAGTCCACATTGATTATTTGCACGGCGTCACACTTTGCTATGCCATAGCATTTTTATCCATAAGATTAGCGGATC CTACCTGACGCTTTTTATCGCAACTCTCTACTGTTTCTCCATACCCGTTTTTTGGGCTAAC [ara promoter]

AAAGAGGAGAAA [RBS]

AGAGAGAGAGAGAGAGAGAGAGCTAAATTAAAAAAAAAACCCAGAAGTGGGAAATCTTCCCCATGAAATAACGGATCCTC TTGCTACTGCTACTACTACTACTACAAACTGTAGCCATTTATATAATTCTATATAATTTTCAACATGGCCACCACCTCTT TAGCTTCCGCTTTCTGCTCGATGAAAGCTGTAATGTTGGCTCGTGATGGCCGGGGCATGAAACCCAGGAGCAGTGATTTG CAGCTGAGGGCGGGAAATGCGCCAACCTCTTTGAAGATGATCAATGGGACCAAGTTCAGTTACACGGAGAGCTTGAAAAG GTTGCCTGACTGGAGCATGCTCTTTGCAGTGATCACAACCATCTTTTCGGCTGCTGAGAAGCAGTGGACCAATCTGGAGT GGAAGCCGAAGCCGAAGCTACCCCAGTTGCTTGATGACCATTTTGGACTGCATGGGTTAGTTTTCAGGCGCACCTTTGCC ATCAGATCTTATGAGGTGGGACCTGACCGCTCCACATCTATACTGGCTGTTATGAATCACATGCAGGAGGCTACACTTAA TCATGCGAAGAGTGTGGGAATCCTAGGAGATGGATTCGGGACGACGCTAGAGATGAGTAAGAGAGATCTGATGTGGGTTG TGAGACGCACGCATGTTGCTGTGGAACGGTACCCTACTTGGGGTGATACTGTAGAAGTAGAGTGCTGGATTGGTGCATCT GGAAATAATGGCATGCGACGTGATTTCCTTGTCCGGGACTGCAAAACAGGCGAAATTCTTACAAGATGTACCAGCCTTTC GGTGCTGATGAATACAAGGACAAGGAGGTTGTCCACAATCCCTGACGAAGTTAGAGGGGAGATAGGGCCTGCATTCATTG ATAATGTGGCTGTCAAGGACGATGAAATTAAGAAACTACAGAAGCTCAATGACAGCACTGCCGATTACATCCAAGGAGGT TTGACTCCTCGATGGAATGATTTGGATGTCAATCAGCATGTGAACAACCTCAAATACGTTGCCTGGGTTTTTGAGACCGT CCCAGACTCCATCTTTGAGAGTCATCATATTTCCAGCTTCACTCTTGAATACAGGAGAGAGTGCACGAGGGATAGCGTGC TGCGGTCCCTGACCACTGTCTCTGGTGGCTCGTCGGAGGCTGGGTTAGTGTGCGATCACTTGCTCCAGCTTGAAGGTGGG TCTGAGGTATTGAGGGCAAGAACAGAGTGGAGGCCTAAGCTTACCGATAGTTTCAGAGGGATTAGTGTGATACCCGCAGA ACCGAGGGTGTAACTAATGAAAGAAGCATCTGTTGAAGTTTCTCCCATGCTGTTCGTGAGGATACTTTTTAGAAGCTGTA GTTTGCATTGCTTGTGCAGAATCATGGTCTGTGGTTTTAGATGTATATAAAAAATAGTCCTGTAGTCATGAAACTTAATA TCAGAAAAATAACTCAATGGGTCAAGGTTATCGAAGTAGTCATTTAAGCTTTGAAATATGTTTTGTATTCCTCGGCTTAA TCTGTAAGCTCTTTCTCTTGCAATAAAGTTCGCCTTTCAAT [BTE CDS]

CTGCTAACAAAGCCCGAAAGGAAGCTGAGTTGGCTGCTGCCACCGCTGAGCAATAACTAGCATAACCCCTTGGGGCCTCT AAACGGGTCTTGAGGGGTTTTTTGCTGAAAGGAGGAACTATATCCGGAT [T7 terminator]

Tactagt [suffix]

AtFatA:

1.ACP Thioesterase AtFatA

Function

Acyl-acyl carrier protein (ACP) thioesterase catalyzes the hydrolysis of acyl-ACP to fatty acids and ACP this reaction removes the coupling between fatty acid biosynthesis and phospholipid biosyntheis,which is a key to obtain free fatty acids. This thioesterase AtFatA can produce fatty acids with 16~18 carbon chain length. In previous researches, thioesterase gene AtFatA can be expressed in E.coil BL21(DE3) and enhance the synthesis of free fatty acids.

Usage and Biology

This part can increase the productivity of free fatty acids. However, this part can not express itself. You should add promoter, RBS and terminator to it or use other parts we offer.

Sequence and Features

Tctagag [prefix]

AATACTAGAAGAGATTCATTCACAGTGTTGAAATTAAAAAACCGAAACTTTCTCGTTTCTTCTTCTTCTTCTCCAATTT TCAAAATTCGAAAAGATGTTGAAGCTTTCGTGTAATGTGACTGATTCTAAGTTACAGAGAAGCTTACTCTTCTTCTCCC ATTCATATCGATCTGATCCGGTGAATTTCATCCGTCGGAGAATTGTCTCTTGTTCTCAGACGAAGAAGACAGGTTTGGT TCCTTTGCGTGCTGTTGTATCTGCTGATCAAGGAAGTGTGGTTCAAGGTTTGGCTACTCTCGCGGATCAGCTCCGATTA GGTAGTTTGACTGAAGATGGTTTATCTTATAAAGAGAAGTTTGTTGTTAGATCTTACGAAGTGGGTAGTAACAAAACCG CTACTGTTGAAACCATTGCTAATCTTTTACAGGAGGTGGGATGTAATCATGCACAAAGTGTTGGTTTTTCGACTGATGG GTTTGCAACAACAACTACTATGAGGAAGTTGCATCTCATTTGGGTTACTGCGAGAATGCATATCGAGATCTATAAGTAC CCTGCTTGGGGTGATGTGGTTGAGATAGAGACTTGGTGTCAGAGTGAAGGAAGGATTGGGACAAGGCGTGATTGGATTC TTAAGGATTCTGTCACTGGTGAAGTCACTGGCCGTGCTACAAGCAAGTGGGTGATGATGAACCAAGACACGAGACGGCT TCAGAAAGTTTCTGATGATGTTCGGGACGAGTACTTGGTCTTCTGTCCTCAAGAACCGAGGTTAGCATTTCCGGAAGAG AATAACAGAAGCTTGAAGAAAATCCCGAAACTCGAAGATCCGGCTCAGTATTCAATGATTGGGCTTAAGCCTAGACGAG CTGATCTCGACATGAACCAGCATGTCAATAATGTCACCTATATTGGATGGGTTCTCGAGAGCATACCACAAGAAATTGT AGACACGCACGAGCTTCAGGTCATAACTCTGGATTATAGAAGAGAATGTCAACAAGACGATGTGGTGGATTCACTCACC ACCACCACCTCTGAAATTGGTGGAACCAATGGCTCTGCCACGTCTGGCACACAGGGCCACAACGATAGCCAGTTCTTGC ACCTCCTGAGGTTGTCTGGAGATGGTCAGGAGATCAACCGCGGGACAACTCTGTGGAGAAAGAAGCCTTCAAGTTAAGA AATAGACAATGTCTTTAGCCATTTTGTTCTCAAGTTTCCATCATCTCAATGAAGATTTCGCTTCACGAGTCTGAGCAGG TCTCCATTTTTTTCTCTTTCAAGTTTGGGTTAGACTAGAGGGAACTGGATTGTTGGAGTATTAATCTTTGTTGAATTTC ATTATGTTTGTTCATGTTTTTGTACAAATTTTGGGGATTTAGCCAAACCCATATCGTCTTTGGTAAAAAAAAAA [AtFatA CDS]

Tactagt [suffix]

2.ACP Thioesterase AtFatA(self-expression system with T7 promoter)

Function

Acyl-acyl carrier protein (ACP) thioesterase catalyzes the hydrolysis of acyl-ACP to fatty acids and ACP this reaction removes the coupling between fatty acid biosynthesis and phospholipid biosyntheis,which is a key to obtain free fatty acids. This thioesterase AtFatA can produce fatty acids with 16~18 carbon chain length. In previous researches, thioesterase gene AtFatA can be expressed in E.coil BL21(DE3) and enhance the synthesis of free fatty acids.

This thioesterase can express itself with a T7 promoter.

Usage and Biology

The RBS and T7 promoter are in front of the AtFatA coding sequence and T7 terminator is after AtFatA. You can only use this part in E.coil BL21(DE3) or choose other parts we offer in other strains.

Sequence and Features

Tctagag [prefix]

GCGGCCGCTTCTAGATAATACGACTCACTATAGG [T7 promoter]

AAAGAGGAGAAA [RBS]

AATACTAGAAGAGATTCATTCACAGTGTTGAAATTAAAAAACCGAAACTTTCTCGTTTCTTCTTCTTCTTCTCCAATTTT CAAAATTCGAAAAGATGTTGAAGCTTTCGTGTAATGTGACTGATTCTAAGTTACAGAGAAGCTTACTCTTCTTCTCCCAT TCATATCGATCTGATCCGGTGAATTTCATCCGTCGGAGAATTGTCTCTTGTTCTCAGACGAAGAAGACAGGTTTGGTTCC TTTGCGTGCTGTTGTATCTGCTGATCAAGGAAGTGTGGTTCAAGGTTTGGCTACTCTCGCGGATCAGCTCCGATTAGGTA GTTTGACTGAAGATGGTTTATCTTATAAAGAGAAGTTTGTTGTTAGATCTTACGAAGTGGGTAGTAACAAAACCGCTACT GTTGAAACCATTGCTAATCTTTTACAGGAGGTGGGATGTAATCATGCACAAAGTGTTGGTTTTTCGACTGATGGGTTTGC AACAACAACTACTATGAGGAAGTTGCATCTCATTTGGGTTACTGCGAGAATGCATATCGAGATCTATAAGTACCCTGCTT GGGGTGATGTGGTTGAGATAGAGACTTGGTGTCAGAGTGAAGGAAGGATTGGGACAAGGCGTGATTGGATTCTTAAGGAT TCTGTCACTGGTGAAGTCACTGGCCGTGCTACAAGCAAGTGGGTGATGATGAACCAAGACACGAGACGGCTTCAGAAAGT TTCTGATGATGTTCGGGACGAGTACTTGGTCTTCTGTCCTCAAGAACCGAGGTTAGCATTTCCGGAAGAGAATAACAGAA GCTTGAAGAAAATCCCGAAACTCGAAGATCCGGCTCAGTATTCAATGATTGGGCTTAAGCCTAGACGAGCTGATCTCGAC ATGAACCAGCATGTCAATAATGTCACCTATATTGGATGGGTTCTCGAGAGCATACCACAAGAAATTGTAGACACGCACGA GCTTCAGGTCATAACTCTGGATTATAGAAGAGAATGTCAACAAGACGATGTGGTGGATTCACTCACCACCACCACCTCTG AAATTGGTGGAACCAATGGCTCTGCCACGTCTGGCACACAGGGCCACAACGATAGCCAGTTCTTGCACCTCCTGAGGTTG TCTGGAGATGGTCAGGAGATCAACCGCGGGACAACTCTGTGGAGAAAGAAGCCTTCAAGTTAAGAAATAGACAATGTCTT TAGCCATTTTGTTCTCAAGTTTCCATCATCTCAATGAAGATTTCGCTTCACGAGTCTGAGCAGGTCTCCATTTTTTTCTC TTTCAAGTTTGGGTTAGACTAGAGGGAACTGGATTGTTGGAGTATTAATCTTTGTTGAATTTCATTATGTTTGTTCATGT TTTTGTACAAATTTTGGGGATTTAGCCAAACCCATATCGTCTTTGGTAAAAAAAAAA [AtFatA CDS]

CTGCTAACAAAGCCCGAAAGGAAGCTGAGTTGGCTGCTGCCACCGCTGAGCAATAACTAGCATAACCCCTTGGGGCCTCT AAACGGGTCTTGAGGGGTTTTTTGCTGAAAGGAGGAACTATATCCGGAT[T7 terminator]

Tactagt [suffix]

3.ACP Thioesterase AtFatA(with a promoter absent)

Function

Acyl-acyl carrier protein (ACP) thioesterase catalyzes the hydrolysis of acyl-ACP to fatty acids and ACP this reaction removes the coupling between fatty acid biosynthesis and phospholipid biosyntheis,which is a key to obtain free fatty acids. This thioesterase AtFatA can produce fatty acids with 16~18 carbon chain length. In previous researches, thioesterase gene AtFatA can be expressed in E.coil BL21(DE3) and enhance the synthesis of free fatty acids.

This thioesterase can not express itself .

Usage and Biology

The RBS is in front of the AtFatA coding sequence and T7 terminator is after AtFatA. You can design your own promoter for this part, but use this part in E.coil BL21(DE3) only or you’d better delete the T7 terminator.

Sequence and Features

Tctagag [prefix]

AAAGAGGAGAAA [RBS]

AATACTAGAAGAGATTCATTCACAGTGTTGAAATTAAAAAACCGAAACTTTCTCGTTTCTTCTTCTTCTTCTCCAATTTT CAAAATTCGAAAAGATGTTGAAGCTTTCGTGTAATGTGACTGATTCTAAGTTACAGAGAAGCTTACTCTTCTTCTCCCAT TCATATCGATCTGATCCGGTGAATTTCATCCGTCGGAGAATTGTCTCTTGTTCTCAGACGAAGAAGACAGGTTTGGTTCC TTTGCGTGCTGTTGTATCTGCTGATCAAGGAAGTGTGGTTCAAGGTTTGGCTACTCTCGCGGATCAGCTCCGATTAGGTA GTTTGACTGAAGATGGTTTATCTTATAAAGAGAAGTTTGTTGTTAGATCTTACGAAGTGGGTAGTAACAAAACCGCTACT GTTGAAACCATTGCTAATCTTTTACAGGAGGTGGGATGTAATCATGCACAAAGTGTTGGTTTTTCGACTGATGGGTTTGC AACAACAACTACTATGAGGAAGTTGCATCTCATTTGGGTTACTGCGAGAATGCATATCGAGATCTATAAGTACCCTGCTT GGGGTGATGTGGTTGAGATAGAGACTTGGTGTCAGAGTGAAGGAAGGATTGGGACAAGGCGTGATTGGATTCTTAAGGAT TCTGTCACTGGTGAAGTCACTGGCCGTGCTACAAGCAAGTGGGTGATGATGAACCAAGACACGAGACGGCTTCAGAAAGT TTCTGATGATGTTCGGGACGAGTACTTGGTCTTCTGTCCTCAAGAACCGAGGTTAGCATTTCCGGAAGAGAATAACAGAA GCTTGAAGAAAATCCCGAAACTCGAAGATCCGGCTCAGTATTCAATGATTGGGCTTAAGCCTAGACGAGCTGATCTCGAC ATGAACCAGCATGTCAATAATGTCACCTATATTGGATGGGTTCTCGAGAGCATACCACAAGAAATTGTAGACACGCACGA GCTTCAGGTCATAACTCTGGATTATAGAAGAGAATGTCAACAAGACGATGTGGTGGATTCACTCACCACCACCACCTCTG AAATTGGTGGAACCAATGGCTCTGCCACGTCTGGCACACAGGGCCACAACGATAGCCAGTTCTTGCACCTCCTGAGGTTG TCTGGAGATGGTCAGGAGATCAACCGCGGGACAACTCTGTGGAGAAAGAAGCCTTCAAGTTAAGAAATAGACAATGTCTT TAGCCATTTTGTTCTCAAGTTTCCATCATCTCAATGAAGATTTCGCTTCACGAGTCTGAGCAGGTCTCCATTTTTTTCTC TTTCAAGTTTGGGTTAGACTAGAGGGAACTGGATTGTTGGAGTATTAATCTTTGTTGAATTTCATTATGTTTGTTCATGT TTTTGTACAAATTTTGGGGATTTAGCCAAACCCATATCGTCTTTGGTAAAAAAAAAA [AtFatA CDS]

CTGCTAACAAAGCCCGAAAGGAAGCTGAGTTGGCTGCTGCCACCGCTGAGCAATAACTAGCATAACCCCTTGGGGCCTCT AAACGGGTCTTGAGGGGTTTTTTGCTGAAAGGAGGAACTATATCCGGAT [T7 terminator]

Tactagt [suffix]

4.ACP Thioesterase AtFatA(regulated-expression system with lac promoter)

Function

Acyl-acyl carrier protein (ACP) thioesterase catalyzes the hydrolysis of acyl-ACP to fatty acids and ACP this reaction removes the coupling between fatty acid biosynthesis and phospholipid biosyntheis,which is a key to obtain free fatty acids.In before study, thioesterase gene AtFatA can express in E.coil BL21(DE3),and increase free fatty acids.

This thioesterase can be expressed in the presence of lactose or IPTG.

Usage and Biology

The RBS and lac promoter are in front of the AtFatA coding sequence and T7 terminator is after AtFatA. We can activate the expression of AtFatA by adding lactose in the culture in the absence of glucose.You can only use this part in E.coil BL21(DE3) because the lac promoter we use is the hybrid of lac promoter and T7 promoter.

Sequence and Features

Tctagag [prefix]

GAAATTAATACGACTCACTATAGGGGAATTGTGAGCGGATAACAATTCCCCACTAGAAATAATTTTGTTTAACTTTAAG [lac promoter]

AAAGAGGAGAAA [RBS]

AATACTAGAAGAGATTCATTCACAGTGTTGAAATTAAAAAACCGAAACTTTCTCGTTTCTTCTTCTTCTTCTCCAATTTT CAAAATTCGAAAAGATGTTGAAGCTTTCGTGTAATGTGACTGATTCTAAGTTACAGAGAAGCTTACTCTTCTTCTCCCAT TCATATCGATCTGATCCGGTGAATTTCATCCGTCGGAGAATTGTCTCTTGTTCTCAGACGAAGAAGACAGGTTTGGTTCC TTTGCGTGCTGTTGTATCTGCTGATCAAGGAAGTGTGGTTCAAGGTTTGGCTACTCTCGCGGATCAGCTCCGATTAGGTA GTTTGACTGAAGATGGTTTATCTTATAAAGAGAAGTTTGTTGTTAGATCTTACGAAGTGGGTAGTAACAAAACCGCTACT GTTGAAACCATTGCTAATCTTTTACAGGAGGTGGGATGTAATCATGCACAAAGTGTTGGTTTTTCGACTGATGGGTTTGC AACAACAACTACTATGAGGAAGTTGCATCTCATTTGGGTTACTGCGAGAATGCATATCGAGATCTATAAGTACCCTGCTT GGGGTGATGTGGTTGAGATAGAGACTTGGTGTCAGAGTGAAGGAAGGATTGGGACAAGGCGTGATTGGATTCTTAAGGAT TCTGTCACTGGTGAAGTCACTGGCCGTGCTACAAGCAAGTGGGTGATGATGAACCAAGACACGAGACGGCTTCAGAAAGT TTCTGATGATGTTCGGGACGAGTACTTGGTCTTCTGTCCTCAAGAACCGAGGTTAGCATTTCCGGAAGAGAATAACAGAA GCTTGAAGAAAATCCCGAAACTCGAAGATCCGGCTCAGTATTCAATGATTGGGCTTAAGCCTAGACGAGCTGATCTCGAC ATGAACCAGCATGTCAATAATGTCACCTATATTGGATGGGTTCTCGAGAGCATACCACAAGAAATTGTAGACACGCACGA GCTTCAGGTCATAACTCTGGATTATAGAAGAGAATGTCAACAAGACGATGTGGTGGATTCACTCACCACCACCACCTCTG AAATTGGTGGAACCAATGGCTCTGCCACGTCTGGCACACAGGGCCACAACGATAGCCAGTTCTTGCACCTCCTGAGGTTG TCTGGAGATGGTCAGGAGATCAACCGCGGGACAACTCTGTGGAGAAAGAAGCCTTCAAGTTAAGAAATAGACAATGTCTT TAGCCATTTTGTTCTCAAGTTTCCATCATCTCAATGAAGATTTCGCTTCACGAGTCTGAGCAGGTCTCCATTTTTTTCTC TTTCAAGTTTGGGTTAGACTAGAGGGAACTGGATTGTTGGAGTATTAATCTTTGTTGAATTTCATTATGTTTGTTCATGT TTTTGTACAAATTTTGGGGATTTAGCCAAACCCATATCGTCTTTGGTAAAAAAAAAA [AtFatA CDS]

CTGCTAACAAAGCCCGAAAGGAAGCTGAGTTGGCTGCTGCCACCGCTGAGCAATAACTAGCATAACCCCTTGGGGCCTCT AAACGGGTCTTGAGGGGTTTTTTGCTGAAAGGAGGAACTATATCCGGAT [T7 terminator]

Tactagt [suffix]

Promoter:

1.FadD Promoter

This part is sensitive to long chain acyl-CoA ,with a FadR binding site.

1. In the absence of long chain acyl-CoA, FadR represses the transcription.

2. In the presence of long chain acyl-CoA, FadR dissociates from the binding site so that the transcription begins.

3. Another promoter named FabB has the same binding site and they will have interactive effect.

Usage and Biology

E.coil uses this promoter to regulate fatty acid metabolism.

Sequence and Features

CGCTTCGCAACCTTTTCGTTGGGTAATTATCAAGCTGGTATGATGAGTTAATATTATGTT

2.FabB Promoter

This part is sensitive to long chain acyl-CoA ,with a FadR binding site.

1. In the absence of long chain acyl-CoA, FadR promote the transcription.

2. In the presence of long chain acyl-CoA, FadR dissociates from the binding site so that the transcription stops.

3. Another promoter named FadD has the same binding site and they will have interactive effect.

Usage and Biology

E.coil uses this promoter to regulate fatty acid metabolism.

Sequence and Features

TTCTCTATTAAATGGCTGATCGGACTTGTTCGGCGTACAAGTGTACGCTATTGTGCATTC

3.Promoter (lacI regulated, T7-lac hybrid)

This part is an inverting regulator sensitive to LacI (BBa_C0010, BBa_C0012, etc.)

The T7-lac promoter is a hybrid regulatory region consisting of the T7 promoter of T7 phage next to LacO. The hybrid design allows for strong promotion that can nevertheless be:

1. repressed by LacI

2. induced by lac or IPTG, showing a high expression level.

3. CAP protein will not influence the expression.

Usage and Biology

Strong promoter. Only E.Coli BL21(DE3) can use this part.

Sequence and Features

GAAATTAATACGACTCACTATAGGGGAATTGTGAGCGGATAACAATTCCCCACTAGAAATAATTTTGTTTAACTTTAAG

4.Pbad promoter

This part contains two protein binding sites. The first binds the CAP protein, which is generally present in E.coli and is asocciated with cell health and availability of glucose. The second binds AraC protein.

• In the absence of AraC protein and CAP protein, this part promotes transcription.

• In the presence of AraC protein and CAP protein, this part inhibits transcription.

Usage and Biology

This is a direct copy of Pbad promoter in E.Coli DH5α ,the natural promoter for the Ara operon.

Sequence and Features

AAACCAATTGTCCATATTGCATCAGACATTGCCGTCACTGCGTCTTTTACTGGCTCTTCTCGCTAACCAAACCGGTAACC CCGCTTATTAAAAGCATTCTGTAACAAAGCGGGACCAAAGCCATGACAAAAACGCGTAACAAAAGTGTCTATAATCACGG CAGAAAAGTCCACATTGATTATTTGCACGGCGTCACACTTTGCTATGCCATAGCATTTTTATCCATAAGATTAGCGGATC CTACCTGACGCTTTTTATCGCAACTCTCTACTGTTTCTCCATACCCGTTTTTTGGGCTAAC

“Traffic light system”

FabB+GFP

This part is sensitive to long chain acyl-CoA ,with a FadR binding site.

• In the absence of long chain acyl-CoA, FadR represses the transcription of GFP.

• In the presence of long chain acyl-CoA, FadR dissociates from the binding site so that the transcription of GFP begins.

• Another promoter named FabB has the same binding site and they will have interactive effect.

Usage and Biology

This part can be used to test the function of FadB. It can also indicates the relative concentration of fatty acid. If you want to use this promoter, choose another part we offer.

Sequence and Features

Tctagag [prefix]

TTCTCTATTAAATGGCTGATCGGACTTGTTCGGCGTACAAGTGTACGCTATTGTGCATTC [FabB promoter]

AAAGAGGAGAAA [RBS]

TACTAGATGCGTAAAGGAGAAGAACTTTTCACTGGAGTTGTCCCAATTCTTGTTGAATTAGATGGTGATGTTAATGGGCA CAAATTTTCTGTCAGTGGAGAGGGTGAAGGTGATGCAACATACGGAAAACTTACCCTTAAATTTATTTGCACTACTGGAA AACTACCTGTTCCATGGCCAACACTTGTCACTACTTTCGGTTATGGTGTTCAATGCTTTGCGAGATACCCAGATCATATG AAACAGCATGACTTTTTCAAGAGTGCCATGCCCGAAGGTTATGTACAGGAAAGAACTATATTTTTCAAAGATGACGGGAA CTACAAGACACGTGCTGAAGTCAAGTTTGAAGGTGATACCCTTGTTAATAGAATCGAGTTAAAAGGTATTGATTTTAAAG AAGATGGAAACATTCTTGGACACAAATTGGAATACAACTATAACTCACACAATGTATACATCATGGCAGACAAACAAAAG AATGGAATCAAAGTTAACTTCAAAATTAGACACAACATTGAAGATGGAAGCGTTCAACTAGCAGACCATTATCAACAAAA TACTCCAATTGGCGATGGCCCTGTCCTTTTACCAGACAACCATTACCTGTCCACACAATCTGCCCTTTCGAAAGATCCCA ACGAAAAGAGAGACCACATGGTCCTTCTTGAGTTTGTAACAGCTGCTGGGATTACACATGGCATGGATGAACTATACAAA TAATAA [GFP]

Tactagt [suffix]

FadD+RFP

This part is sensitive to long chain acyl-CoA ,with a FadR binding site.

• In the absence of long chain acyl-CoA, FadR represses the transcription of RFP.

• In the presence of long chain acyl-CoA, FadR dissociates from the binding site so that the transcription of RFP begins.

• Another promoter named FabB has the same binding site and they will have interactive effect.

Usage and Biology

This part can be used to test the function of FadD. It can also indicates the relative concentration of fatty acid. If you want to use this promoter, choose another part we offer.

Sequence and Features

Tctagag [prefix]

CGCTTCGCAACCTTTTCGTTGGGTAATTATCAAGCTGGTATGATGAGTTAATATTATGTT [FadD promoter]

AAAGAGGAGAAA [RBS]

CACATGGCATGGATGAACTATACAAATAATAATACTAGAGAAAGAGGAGAAATACTAGATGGCTTCCTCCGAAGACGTTA TCAAAGAGTTCATGCGTTTCAAAGTTCGTATGGAAGGTTCCGTTAACGGTCACGAGTTCGAAATCGAAGGTGAAGGTGAA GGTCGTCCGTACGAAGGTACCCAGACCGCTAAACTGAAAGTTACCAAAGGTGGTCCGCTGCCGTTCGCTTGGGACATCCT GTCCCCGCAGTTCCAGTACGGTTCCAAAGCTTACGTTAAACACCCGGCTGACATCCCGGACTACCTGAAACTGTCCTTCC CGGAAGGTTTCAAATGGGAACGTGTTATGAACTTCGAAGACGGTGGTGTTGTTACCGTTACCCAGGACTCCTCCCTGCAA GACGGTGAGTTCATCTACAAAGTTAAACTGCGTGGTACCAACTTCCCGTCCGACGGTCCGGTTATGCAGAAAAAAACCAT GGGTTGGGAAGCTTCCACCGAACGTATGTACCCGGAAGACGGTGCTCTGAAAGGTGAAATCAAAATGCGTCTGAAACTGA AAGACGGTGGTCACTACGACGCTGAAGTTAAAACCACCTACATGGCTAAAAAACCGGTTCAGCTGCCGGGTGCTTACAAA ACCGACATCAAACTGGACATCACCTCCCACAACGAAGACTACACCATCGTTGAACAGTACGAACGTGCTGAAGGTCGTCA CTCCACCGGTGCTTAATAACGCTGATAGTGCTAGTGTAGATCGCTACTAGAGCCAGGCATCAAATAAAACGAAAGGCTCA GTCGAAAGACTGGGCCTTTCGTTTTATCTGTTGTTTGTCGGTGAACGCTCTCTACTAGAGTCACACTGGCTCACCTTCGG GTGGGCCTTTC [RFP]

Tactagt [suffix]

FabB regulated GFP and FadD regulated RFP

Function

FabB promoter and FabD promoter reacts differently to the changing in concentration of fatty acids. We activate the expression of GFP and RFP by FabB promoter and FabD promoter respectively. When fatty acid concentration fluctuates, the changing in the expression of GFP and RFP tells us the fluctuation of fatty acid concentration.

Usage and Biology

This part functions as a biosensor. In the abundance of fatty acid, the red fluorescence is bright. On the contrary, in the absence of fatty acid, the green fluorescence is bright.

Sequence and Features

Tctagag [prefix]

CGCTTCGCAACCTTTTCGTTGGGTAATTATCAAGCTGGTATGATGAGTTAATATTATGTT [FadD promoter]

AAAGAGGAGAAA [RBS]

CACATGGCATGGATGAACTATACAAATAATAATACTAGAGAAAGAGGAGAAATACTAGATGGCTTCCTCCGAAGACGTTA TCAAAGAGTTCATGCGTTTCAAAGTTCGTATGGAAGGTTCCGTTAACGGTCACGAGTTCGAAATCGAAGGTGAAGGTGAA GGTCGTCCGTACGAAGGTACCCAGACCGCTAAACTGAAAGTTACCAAAGGTGGTCCGCTGCCGTTCGCTTGGGACATCCT GTCCCCGCAGTTCCAGTACGGTTCCAAAGCTTACGTTAAACACCCGGCTGACATCCCGGACTACCTGAAACTGTCCTTCC CGGAAGGTTTCAAATGGGAACGTGTTATGAACTTCGAAGACGGTGGTGTTGTTACCGTTACCCAGGACTCCTCCCTGCAA GACGGTGAGTTCATCTACAAAGTTAAACTGCGTGGTACCAACTTCCCGTCCGACGGTCCGGTTATGCAGAAAAAAACCAT GGGTTGGGAAGCTTCCACCGAACGTATGTACCCGGAAGACGGTGCTCTGAAAGGTGAAATCAAAATGCGTCTGAAACTGA AAGACGGTGGTCACTACGACGCTGAAGTTAAAACCACCTACATGGCTAAAAAACCGGTTCAGCTGCCGGGTGCTTACAAA ACCGACATCAAACTGGACATCACCTCCCACAACGAAGACTACACCATCGTTGAACAGTACGAACGTGCTGAAGGTCGTCA CTCCACCGGTGCTTAATAACGCTGATAGTGCTAGTGTAGATCGCTACTAGAGCCAGGCATCAAATAAAACGAAAGGCTCA GTCGAAAGACTGGGCCTTTCGTTTTATCTGTTGTTTGTCGGTGAACGCTCTCTACTAGAGTCACACTGGCTCACCTTCGG GTGGGCCTTTC [RFP]

CTGCTAACAAAGCCCGAAAGGAAGCTGAGTTGGCTGCTGCCACCGCTGAGCAATAACTAGCATAACCCCTTGGGGCCTCT AAACGGGTCTTGAGGGGTTTTTTGCTGAAAGGAGGAACTATATCCGGATCTGCTAACAAAGCCCGAAAGGAAGCTGAGTT GGCTGCTGCCACCGCTGAGCAATAACTAGCATAACCCCTTGGGGCCTCTAAACGGGTCTTGAGGGGTTTTTTGCTGAAAG GAGGAACTATATCCGGAT [double T7 terminator]

TTCTCTATTAAATGGCTGATCGGACTTGTTCGGCGTACAAGTGTACGCTATTGTGCATTC [FabB promoter]

AAAGAGGAGAAA [RBS]

TACTAGATGCGTAAAGGAGAAGAACTTTTCACTGGAGTTGTCCCAATTCTTGTTGAATTAGATGGTGATGTTAATGGGCA CAAATTTTCTGTCAGTGGAGAGGGTGAAGGTGATGCAACATACGGAAAACTTACCCTTAAATTTATTTGCACTACTGGAA AACTACCTGTTCCATGGCCAACACTTGTCACTACTTTCGGTTATGGTGTTCAATGCTTTGCGAGATACCCAGATCATATG AAACAGCATGACTTTTTCAAGAGTGCCATGCCCGAAGGTTATGTACAGGAAAGAACTATATTTTTCAAAGATGACGGGAA CTACAAGACACGTGCTGAAGTCAAGTTTGAAGGTGATACCCTTGTTAATAGAATCGAGTTAAAAGGTATTGATTTTAAAG AAGATGGAAACATTCTTGGACACAAATTGGAATACAACTATAACTCACACAATGTATACATCATGGCAGACAAACAAAAG AATGGAATCAAAGTTAACTTCAAAATTAGACACAACATTGAAGATGGAAGCGTTCAACTAGCAGACCATTATCAACAAAA TACTCCAATTGGCGATGGCCCTGTCCTTTTACCAGACAACCATTACCTGTCCACACAATCTGCCCTTTCGAAAGATCCCA ACGAAAAGAGAGACCACATGGTCCTTCTTGAGTTTGTAACAGCTGCTGGGATTACACATGGCATGGATGAACTATACAAA TAATAA [GFP]

CTGCTAACAAAGCCCGAAAGGAAGCTGAGTTGGCTGCTGCCACCGCTGAGCAATAACTAGCATAACCCCTTGGGGCCTCT AAACGGGTCTTGAGGGGTTTTTTGCTGAAAGGAGGAACTATATCCGGATCTGCTAACAAAGCCCGAAAGGAAGCTGAGTT GGCTGCTGCCACCGCTGAGCAATAACTAGCATAACCCCTTGGGGCCTCTAAACGGGTCTTGAGGGGTTTTTTGCTGAAAG GAGGAACTATATCCGGAT [double T7 terminator]

Tactagt [suffix]