Team:Hong Kong UCCKE/Composite Part
Composite Parts
BBa_K3077000
The part BBa_K3077000 is a composite part of the periplasmic α-amylase gene encoded as malS in the E. coli. The genomic sequence of the malS gene is referenced from BBa_K1418020[1] registered by BBa_K1418020 registered by Team Utah State in 2014. Team Utah State removed the native ribosome binding site and the predicted cleavage signal peptide of BBa_K523001[2] in 2014, forming BBa_K1418020. Our teammates further designed it to be composite part BBa_K3077000 by adding a lac promoter R0010[3], strong RBS B0034[4], 10x-Histidine tag K844000[5] and double terminator B0015[6].
Fig.1 Pathway of BBa_K3077000
R0010 is a conditional promoter that promotes transcription when it binds to CAP protein in the absence of Lac repressor, which can be maintained by the presence of IPTG and low glucose level.This can prevent overexpression and keep the cell healthy. B0034, a strong RBS, to achieve a higher expression level. A 10X His-tag, to allow protein purification, is fused at the N terminus of the lipase gene. B0015 is a double terminator combining B0010 and B0012, and is one of the most commonly used and reliable terminator in the iGEM catalog.
The gene BBa_K523001 is the Periplasmic alpha-amylase encoded as malS in E. coli. malS is a hydrolase responsible for degrading amylose and long maltodextrins with at least 3 glucose residues, including starch and glycogen, yielding smaller sugars with even numbers of glucose residues[7]. It recognizes substrates from the non-reducing end and hydrolyses the α-1,4 glycosidic linkages between each glucose monomer[7].
Enzymatic Reaction[8]:
a 1,4-α-D-glucan[periplasm] + n H2O[periplasm] → a 1,4-α-D-glucan[periplasm] + maltohexaose[periplasm]
MalS is thought to provide a growth advantage by degrading periplasmic maltodextrins between 7 and 15 glucose units in length[7]. Since only maltooligosaccharides up to a chain length of 6 glucose units are actively transported through the cytoplasmic membrane via the membrane-bound complex of three proteins, MalF, MalG, and MalK, longer maltooligosaccharides must first be degraded by the MalS protein[7]. It is a member of the maltose regulon, and therefore is under the regulatory control of the MalT protein, the gene activator of the Escherichia coli maltose system[7]. As it is a periplasmic gene, it is not accessible to its substrate directly. Fortunately, in Escherichia coli, maltodextrins enter the periplasm pref-erentially via the outer membrane porin LamB [18].
The activity of malS is maximum at 22℃, slightly reduced to 93% from 37℃ to 42℃, and completely lost at 61 °C[9]. It’s optimum pH is 8-8.5 and is completely denatured at pH 5[18].
The plasmid of composite part BBa_K3077000 failed to be ordered from IDT, so related assays can’t be carried out to test the effectiveness of the design.
BBa_K3077100
This part BBa_K3077100[10], is a composite part of the cytoplasmic esterase encoded as lip8 in Pseudomonas aeruginosa. The genomic sequence of the lip8 gene is referenced from [11]. The lip8 gene, registered as BBa_K3077101[12], is designed to be the composite part BBa_K3077100 by adding lac promoter R0010[3], strong RBS B0034[4], 10x-Histidine tag K844000[5] and double terminator B0015[8].
Fig.2 Pathway of BBa_K3077100
R0010 is a conditional promoter that promotes transcription when it binds to CAP protein in the absence of Lac repressor, which can be maintained by the presence of IPTG and low glucose level. This can prevent overexpression and keep the cell healthy. This can prevent overexpression of the cell and keep it healthy. B0034, a strong RBS, to achieve a higher expression level. A 10X His-tag, to allow protein purification, is fused at the N terminus of the lipase gene. B0015 is a double terminator combining B0010 and B0012, and is one of the most commonly used and reliable terminator in the iGEM catalog.
The gene BBa_K3077101 is a cytoplasmic esterase encoded as lip8 in Pseudomonas aeruginosa. Lip8 is an esterase that catalyzes both the hydrolysis and synthesis of ester bonds. It is also a biocatalyst for interesterification, alcoholysis, acidolysis, and aminolysis.[11] It showed higher activities against short-chain fatty acid methyl esters compared with activities against various triacylglycerols and long-chain fatty acid methyl esters: Among triacylglycerols with different fatty acids, Lip8 was found to have the highest activity against triacetin (C2). While methyl acetate (C2), methyl propionate (C3), and methyl butyrate (C4) are the substrates with higher hydrolytic activity among different fatty acid methyl esters. Among four Natural oils, Lip8 showed highest activity against tung oil, whose principal fatty acid component is eleostearic acid (C18: 3)[11].
This plasmid containing composite part BBa_K3077100 is ordered and synthesized by IDT. The ordered plasmid is transformed in E.coli and is verified through colony PCR with prefix forward and suffix reverse. We tested its catalytic activity as an esterase through the lipid hydrolysis assay.
BBa_K3077200
The part BBa_K3077200[13] is a composite part of the new heterologous lipase encoded as lipIAF5-2. The genomic sequence of the lipIAF5-2 gene is referenced from ENA (Gene ID EU660533)[14]. The lipIAF5-2 gene, registered as BBa_K3077202[2], is designed to be the composite part by adding a constitutive promoter J23111[15], strong RBS B0034[4] and double terminator B0015[8].
Fig.3 Pathway of BBa_K3077200
J23111 is a constitutive promoter that allows continual expression of the gene, it is a consensus promoter sequence and the strongest member of the family, so it can promote a high rate of gene expression. B0034, a strong RBS, to achieve a higher expression level. B0015 is a double terminator combining B0010 and B0012, and is one of the most commonly used and reliable terminator in the iGEM catalog.
The gene BBa_K3077202 is a new heterologous lipase identified by a metagenome library encoded as lipIAF5-2. It is a polypeptide of 308 amino acids with a molecular mass of 32.6 kDa[16]. It is a newly discovered novel gene sequence that showed no more than 52% identity with other lipases[16].
The lipase showed higher activity with long-length acyl chains, showing maximal activity with p-NPM (C14) and about 70% with p-NPM (C16) and p-NPM (C18)[16]. It is extracellular and is secreted out of the cell[16].
Being a true lipase, it is able to efficiently synthesize short chain esters which produces sweet-smelling aromas by transesterification and esterification reactions in organic media. It showed good affinity toward glyceryl trioctanoate and the highest conversion yields were obtained for the transesterification of glyceryl triacetate with methanol. [17]
This plasmid containing composite part BBa_K3077200 is ordered and synthesized by IDT. The plasmid is transformed in E.coli. However, we don’t have sufficient time to verify the clone with Colony PCR as planned.
[1]: BBa_K1418020
[2]: BBa_K523001
[3]: BBa_R0010
[4]: BBa_B0034
[5]: BBa_K844000
[6]: BBa_B0015
[7]: Freundlieb S, Boos W.(1986) a-Amylase of Escherichia coli, Mapping and Cloning of the Structural Gene, mal& and Identification of Its Product as a Periplasmic Protein*. J Bill Chem.261(6):2946-53.
[8]: https://ecocyc.org/ECOLI/NEW-IMAGE?type=REACTION&object=RXN0-5181
[9]: Spiess C1, Happersberger HP, Glocker MO, Spiess E, Rippe K, Ehrmann M.Biochemical characterization and mass spectrometric disulfide bond mapping of periplasmic alpha-amylase MalS of Escherichia coli.J Biol Chem. 1997 Aug 29;272(35):22125-33.
[10]: BBa_K3077100
[11]: Ogino H1, Mimitsuka T, Muto T, Matsumura M, Yasuda M, Ishimi K, Ishikawa H.Cloning, expression, and characterization of a lipolytic enzyme gene (lip8) from Pseudomonas aeruginosa LST-03. J Mol Microbiol Biotechnol. 2004;7(4):212-23.
[12]: BBa K3077101
[13]: BBa_K3077200
[14]: https://www.ebi.ac.uk/ena/data/view/EU660533
[15]: BBa_J23111
[16]: Meilleur C., Hupe J.F., Juteau P., Shareck F(2009). "Isolation and characterization of a new alkali-thermostable lipase cloned from a metagenomic library."J. Ind. Microbiol. Biotechnol. 36:853-861(2009)
[17]: Brault G, Shareck F, Hurtubise Y, Lépine F, Doucet N (2014) Short-Chain Flavor Ester Synthesis in Organic Media by an E. coli Whole-Cell Biocatalyst Expressing a Newly Characterized Heterologous Lipase. PLoS ONE 9(3): e91872. https://doi.org/10.1371/journal.pone.0091872
[18]Spiess, C., Happersberger, H. P., Glocker, M. O., Spiess, E., Rippe, K., & Ehrmann, M. (1997). Biochemical Characterization and Mass Spectrometric Disulfide Bond Mapping of Periplasmic α-Amylase MalS ofEscherichia coli. Journal of Biological Chemistry, 272(35), 22125–22133. doi:10.1074/jbc.272.35.22125