Introduction

In case of a low yield we want a targeted secretion only near our (that we want to coat with silk). In order to achieve this we want to have a bacillus that will move towards heat. If the implant is heated it will attract our silk secreting bacillus.

Thermal control of fatty acid synthesis.

In order to maintain the fluidity of the cell membrane when the environmental temperature is decreasing, B. subtilis (among other bacteria) adapts the membrane by increasing the fraction of unsaturated phospholipids acyl chains.

The desaturation of the membrane starts with the membrane protein, DesK. DesK senses temperature of its environment and when the temperature is <30 °C, DesK autophosphorylates its conserved histidine. Sequentially the phosphoryl group is transferred to the aspartate residue in desR that activates the promoter of des. The gene des is translated into a fatty acid desaturase (Δ5-Des), that changes the fluidity of the membrane by introducing double bonds into pre-existing saturated fatty acyl chains.

The promoter activity of des

(a)Pattern of Pdes-lacZ expression on a temperature downshift. B. subtilis AKP3 cells were grown at 37 °C to an optical density of 0.4 at 525 nm and then divided into two fractions. The first was transferred to 25 °C (●) and the second was kept at 37 °C (○). (b) Pattern of Pdes-lacZ expression in a des‾ background. B. subtilis AKP4 cells were grown at 37 °C to an optical density of 0.4 at 525 nm and then divided into two fractions. One fraction was transferred to 25 °C (●) while the other was kept at 37 °C (○). (c). Effect of exogenous fatty acids on Pdes-lacZ expression pattern. B. subtilis AKP4 cells were grown at 37 °C to an optical density of 0.4 at 525 nm and then divided into two fractions. Each fraction was supplemented with palmitic (●) or oleic acid (■) and growth was continued at 25 °C. (d) Effect of desKR disruption on Pdes-lacZ expression. B. subtilis AKP21 cells were grown at 37 °C to an optical density of 0.4 at 525 nm and then divided into two fractions. One of the fractions was transferred to 25 °C (●) and the other one was kept at 37 °C (○). Optical density at 525 nm (inserts) and β-galactosidase specific activity were determined at the indicated times (a, b, c, or d).

Motility

Bacterial movement is based on flagella (tail like structures) and utilizes a counter-clockwise (CWW) and clockwise (CW) motion. When the flagella turn CCW they gather in one area resulting in bacteria that move straight. When the flagella move CW they disperse all over the cell membrane, resulting in the bacteria tumbling in random directions. When bacteria sense an attractant the flagellar motion will turn CCW, when the concentration of the attractant reduces the flagella will turn CW.

The receptor is displayed in the figure on the page below. The letters are all Che proteins, with this cascade of proteins motility is coordinated. When a attractant is bound to the receptor, CheA phosphorylizes CheY into CheY-P. CheY-P causes the CCW motility in the flagella, it also causes (by forming a CheY-p/CheC complex) that CheD is pulled off the receptor. CheD and CheC forms also a complex which dephosphorylizes CheY-p into CheY thus resetting the receptor.

The principle

CheY is a mayor factor in spinning the flagella CCW. When cheY is absent, cells are significant less motile[ref]. Because the promoter of des is active at low temperatures (25 °C) we placed cheY under control of the promoter of des (figure 2).

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