Difference between revisions of "Part:BBa K2924051"

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Leaderless 'TesA

Rhamnose-inducible promoter (similar to BBa_K914003) and RBS* (BBa_K2924009¹) expressing the thioesterase tesA (BBa_K1472601)and a double terminator (BBa_B0015).

Usage and Biology

This part contains the rhamnose-inducible promoter P_rha described by Kelly et al. (2018)^-1 which was further modified by Behle et al. (2019)⁰, and is similar to (BBa_K914003), the RBS* (BBa_K2924009)¹, a thioesterase from Escherichia coli (BBa_K1472601) and a double terminator (BBa_B0015).

In general, all basic parts used here (with the exception of ‘TesA) were optimized for Synechocystis sp. PCC 6803, but function efficiently in both E. coli and Synechocystis sp..

This thioesterase lacks a leader peptide sequence, which is the N-terminal 26-amino acid signal peptide sequence. ’TesA is located in the cytosol of E.coli, where it hydrolyzes the bond of fatty acyl to acyl-carrier protein (ACP) or Coenzyme A (CoA). This results in a free fatty acids and either ACP or CoA.

Background

Fig. 1: Structural formula of hexadecanoic acid. Gray spheres represent a carbon atom, red spheres represent oxygen atom and the white spheres represent hydrogen atoms.

Fig. 2: Structural formula of octadecanoic acid. Gray spheres represent a carbon atom, red spheres represent oxygen atom and the white spheres represent hydrogen atoms.

Fatty acids are long aliphatic chained carboxylic acids, which can be saturated or unsaturated. They have mostly an even number of carbon atoms from 4 to 28. Hexadecanoic acid (Fig. 1) is a saturated long-chain fatty acid, which contains 16 carbon-atoms and is also called palmitic acid (C16:0). It has a white or colorless crystalline form and a slightly characteristic odor². It has a molecular weight of 256.42 g/mol.

Octadecanoic acid (Fig. 2) is a saturated long-chain fatty acid, which contains 18 carbon-atoms and is also called stearic acid (C18:0). It is a white or slightly yellow, wax-like solid with mild odor³. It has a molecular weight of 284.48 g/mol.

Usage of palmitic and stearic acid

The long-chain fatty acid palmitic acid (C16:0) is found naturally in human milk, in cow's milk fat for triglycerides or in vegetable oils and it is commonly used in infant formulas⁴. It is the most abundant fatty acid in cow’s milk⁵.

Palmitic acid (C16:0) is an important chemical, which is used to make soaps or cosmetics agents, lubricating oils, waterproofing materials, food additives and other chemicals².

Stearic acid (C18:0) is also found naturally in animal milk and vegetable oils³ and used in manufacturing of different pharmaceuticals, cosmetics, soaps, candles, food packaging, modeling compounds and other chemicals. It is found sometimes in pesticides³.

Moreover, they play an important role for the intracellular biological functions. They mainly serve as source of metabolic energy or as substrates for the cell membrane biosynthesis. Another function is serving as precursor, such as prostaglandins (PGs), leukotrienes and more, for different signaling pathways⁵. It has been shown that saturated fatty acids, such as palmitic and stearic acid (C16:0; C18:0), induce apoptosis of human granulosa cells, which could be used for further medical investigations⁵. Both fatty acids are used for special dietary to increase the milk fat yield and as an energy source for milk production⁶.

Sequence and Features

Characterization

First, the inducible promoter, as well as the terminator with Gibson overhangs were fused to ‘tesA via overlap extension PCR. This PCR product was used for Gibson assembly to ligate it into the pSNDY backbone. The conjugative shuttle vector pSNDY is a pSHDY⁷ derivative, encoding a nourseothricin resistance instead of spectinomycin, and is a broad-host-range vector able to self-replicate in E. coli, as well as to be transferred to other hosts such as cyanobacteria via conjugation.

Fig. 3: Transformants differ in morphology. <i>E. coli BL21 control transformation (left) and E. coli BL21 transformed with pSNDY containing P_rha:’tesA (right).</i>

After transformation of the P_rha:’tesA construct, the transformants of E. coli BL21 seem to differ in morphology (Figure 3).