Parts Human Practices Safety Model Project Team Home

Overview

Acetate production

Oleate consumption

Coexpression of acetate production and β-oxidation pathways

Inhibitory regulator

Prediction of overall performance of acetate production

Biosafety

Degradation-promoting tag

Overview

Here we present the results of our works. Every module in the primary design has been validated experimentally. According to these results, SLIM can not only consume a considerable amount of fatty acids, but also overproduce acetic acid with a remarkable efficiency when activated, which is regulated by the digestion sensor. Our results also suggest that SLIM is reliable in biosafety, being both escape-proof and controllable in use. Furthermore, an advanced version of SLIM has been proposed and is still under tests. In this version, a bilateral switch is included as an alternative regulator, saving unnecessary overexpression of enzymes when not needed and increasing its potential for further modification.

Acetate production

As shown in the chart, by overexpressing phosphotransacetylase (PTA) and acetokinase (ACK), we successfully lifted the yield of acetic acid by ~10 fold 2 hours after induction and maintained it afterwards.

Through our previous investigation we learned that the interval between the end of digestion and the next meal was between 2 – 4 hours. Considering the movement of chyme inside intestine, the actual effect time of acetate overproduction would be longer than that. Moreover, in the intestine, as acetate being absorbed over time, there would be less product inhibition than in flask, ensuring a production rate maintained at a high level. As a result, this overproduction fairly well met our expectation and need.

Fig. 1 Western blot analysis of ACK and PTA.

Fig. 2 Acetate production overtime. The SLIM overproduces about tenfold acetate within and after 2 hours, which approximately equals to time of limosis.

Oleate consumption

We measured the enhancement of oleate consumption when the two enzymes showing most potential in our model, fatty acyl-CoA synthetase (FadD) or acyl-CoA dehydrogenase (FadE), was overexpressed respectively. Oleic acid, as a fatty acid with a general high content in all animal and vegetable oils in the form of glycerides, is used in our experiment to test the general consumption of higher fatty acids.

As shown in Fig. 3, microbe that FadD consumes more than twice as much oleate as control group does when overexpressed, while FadE also shows an effect although with less efficiency.

Fig. 3 Oleate consumption overtime. A: Microbe that overexpress FadD consumes twofold acetate compared to wild type; B: The consumption of acetate by microbe that overexpress FadE is far less than fadD.

Coexpression of acetate production and β-oxidation pathways

β-oxidation of higher fatty acids yields acetyl-CoA which is the substrate of acetate production. Therefore, it is presumable that the coexpression of both pathways shall further enhance the overproduction of acetic acid.

To put this theory into test, we constructed a vector containing pta, ack and fadD. The vector was transferred into a strain, of which the ability to produce acetate was compared to that of the strain that overexpresses only PTA and ACK.

As our result shows, the induction of β-oxidation pathway did enhance acetate production to a further extend by nearly 50%. Although in the integrated system the two pathways are to be expressed at different times, this further enhancement should remain to some degree.

Fig. 4 Acetate production enhanced by FadD overexpression.

Inhibitory regulator

First, to test the threshold of rpoH P5 promoter, we induced the strain with a gradient of glucose concentration. After correction with OD600, the result shows a remarkable difference in fluorescence intensity between bacteria induced with 0.01% and 0.05% glucose and a glucose concentration higher than 0.05% did not further repress fluorescence intensity, indicating a threshold of rpoH P5 promoter between 0.01% and 0.05%.

Combining the curve of glucose concentration inside intestine during digestion simulated through modelling, it is concluded that the sensor can function for as long as two hours. Consequently, the activated time for β-oxidation and acetate production should be both around two hours, leaving enough time for both pathways to function.

Fig. 5 Effects of glucose on the level of rpoH P5 sensitivity. A: Analysis of rpoH sensitivity to different glucose concentrations (0.01%, 0.05%, 0.1% and 1%). B: Intestinal glucose concentration simulation model shows that rpoH P5 promoter respond to slight changes in glucose concentration inside intestine as expected.

Prediction of overall performance of acetate production

IBy experiments we can know that at a low population density, our engineered bacteria produce 0.0213g/mL acetic acid per cell in 45 minutes. In the test of inhibitory promoter rpoH P5, the promoter inhibits production of acetic acid for 2 hours per meal, leaving 18 hours for acetate producing inside small intestine. Meanwhile, production of acetate inside large intestine is activated all day. Considering bacteria density inside intestine ^[1], the accumulated yield of acetic acid in a day would be 24g, if 1% of intestinal microbe is substituted by the engineered bacteria. Compared with the amount of acetic acid used in clinical trials (1.5g/day) ^[2], our acetate overproduction pathway produces more than acetate.

On the other hand, the beta-oxidation pathway is constantly on, yet its substrate is provided only during digestion for a total amount of 6 hours a day.

Biosafety

Induced suicide

Induced suicide module aims to terminate the engineered bacteria inside intestine with induction of L-arabinose whenever needed. Two different concentrations of arabinose were used in our experiment. As displayed below, after induction, the experimental groups showed a sharp drop in survival rate. Otherwise, the OD600 of the culture remained constant, confirming our presumption that MazF, as a cell toxin, does not lyse the bacteria, and is therefore harmless to native intestinal microbes.

Fig. 6 Suicide of microbe induced by L-arabinose. A: Living cells are counted over time after induction, showing a significant decrease under the effect of MazF; B: OD600 proof to be similar among control and experimental groups, which proves the induced suicide does not cause cell lysis.

Kill switch

As shown in the graph, bacteria cultured at room temperature showed a significantly lower survival rate than those cultured at body temperature, indicating our kill switch worked as expected.

Fig. 7 Kill switch triggered by cold temperature. A: Cell density of engineered bacteria at 27℃ is far lower than same strain cultured at 37 ℃ and control groups; B: OD600 is similar between control and experimental groups, which proves the kill switch does not cause cell lysis.

Degradation-promoting tag

Some modules in our project call for efficient degradation of protein, such as recombination directionality factor (RDF) in recombinase system and antitoxin RelB in kill switch. To verify the validity of RepA, a short peptide that induces degradation of protein using connatural E. coli machinery, we tested the efficiency of RepA using GFP as an indicator.

Validation of RepA

As the graph shows, upon removal of inducer, GFP with and without tag showed a remarkable difference in behavior. Average fluorescence intensity of bacteria expressing tag-free gfp roughly remained at high level, while those expressing GFP with tag showed a rapid decline in average fluorescence intensity. These results indicate that degradation tag RepA is valid enough for our project.

Fig. 8 Function of RepA validated by GFP. The fluorescence intensity drops at a rapid speed after removing inducer, indicating the RepA boosts the degradation of GFP.

Antitoxin improved by addition of RepA

As mentioned before, the decline of RelB, the antitoxin, plays a key part in module kill switch. The insufficiency of RelB in a cold temperature causes death of bacteria that escape from human body, preventing contamination of environment. Therefore, the degradation rate of RelB is determinative to the performance of the part.

After validating the kill switch, we further improved it by adding the degradation-promoting tag RepA to the N-terminal of RelB. As is shown in Fig. 9, the improved system shows a notable better function.

Fig. 9 Comparison between kill switches before and after improvement. A: growth curve measured by dilution and counting cell-forming units. B: escape rates of microbe

Reference

[1] Sender R, Fuchs S, Milo R. Revised Estimates for the Number of Human and Bacteria Cells in the Body. PLoS Biol. 2016 Aug 19 14(8):e1002533. doi: 10.1371/journal.pbio.1002533.

[2] Kondo T , Kishi M , Fushimi T , et al. Vinegar Intake Reduces Body Weight, Body Fat Mass, and Serum Triglyceride Levels in Obese Japanese Subjects[J]. Bioscience, Biotechnology and Biochemistry, 2009, 73(8):1837-1843.

Team:BNU-China/Demonstrate