Team:BNU-China/Experiments

Acetate production

Higher fatty acids consumption

Enhancement of acetate production by β-oxidation

Bilateral switch

Degradation tag

Digestion sensors

Induced suicide

Kill switch

Acetate production


To test the ability of our circuit to overproduce acetate, a pathway is constructed as follows: the coding sequence of phosphotransacetylase (PTA) and acetokinase (ACK) downstream of respective RBSs are arranged tandemly as a polycistron under control of a lac promoter. Upon induction, pta and ack get expressed and thereby initiates overproduction of acetic acid. A strain containing a vector with same backbone and irrelevant inserted gene is used as control.

Both experimental and control groups are cultured in 30 mL of LB-ampicillin medium at 37℃ for 5 hours till their OD600 reach around 0.2. Then the bacteria are collected by centrifugation and resuspended with LB-ampicillin containing 5 mM IPTG for induction. Samples are taken at 0 hr, 2 hr and 4 hr after induction and tested using Megazyme acetic acid assay kit.

Higher fatty acids consumption


Various higher fatty acids exist in human diet, and their proportion differ from type to type of food, depending on kinds of fat contained. It is therefore impossible to blend a mixture that accurately represents the ingredient of digestive product of all kinds of fat. Nevertheless, some higher fatty acids have a generally steady and relatively high content in most kinds of fat, oleate for example, that allow us to test relative general consumption of higher fatty acids.

In our experiment, experimental control groups are cultured in LB-ampicillin medium overnight before being diluted with equal amount of LB-ampicillin medium containing 400 mM sodium oleate, making the final concentration of oleate 200 mM. Then both groups are induced with 5 mM IPTG and sampled at 0 hr, 2 hr and 4 hr. Oleate concentration of the samples are tested using Shuangying FFA ELISA kit.

Enhancement of acetate production by β-oxidation


As has been mentioned before, overproduction of acetic acid should be boosted by β-oxidation, which produces acetyl-CoA, the important substrate of the former pathway. But how does the theory work in reality? A pretest is carried out as follows.

The enzymes for two pathways are coexpressed in a same strain. The strain is cultured before centrifuged and resuspended with LB-ampicillin containing 200 mM oleate and 5 mM IPTG. Samples are taken at 0 hr, 2 hr and 4 hr after induction and tested for their content of acetate and higher fatty acids.

Bilateral switch


The bilateral switch in our circuit is made possible by a recombinase system. Two activities of integrase are involved in this system. One of these activities, called integrase activity flips the sequence between recognition sites attB and attP, converting the two sites to attL and attR in the absence of its recombination directionality factor (RDF); the other activity, called excisionase activity, in turn, flips the sequence back, reforming sites attB and attP in the presence of RDF. Consequently, the experiments are carried out as follows.

1)First, the two activities of integrase are tested respectively. In each test, an expressor and a reporter plasmid are constructed using backbones with different antibiotic resistance markers (ampR and cmR). For integrase activity, we put integrase gene (int) under control of an arabinose-induced promoted in expressor plasmid and inverted GFP gene (gfp) flanked by attB and attP sites downstream of a constitutive promoter, so that upon induction, expressed orients gfp in the way such that the latter gets to express. The plasmids for excisionase activity testing are designed in a similar way. The two plasmids are subsequently transferred into a same strain, which serves as experimental group. Positive control group contains an expressor plasmid and a “reporter” plasmid that constantly expresses GFP.

Both experimental and positive control groups are cultured in test tubes containing LB-ampicillin/chloramphenicol medium for 5 hr before induction. Cells are centrifuged and medium refreshed every hour to maintain a steady arabinose level. Every time after resuspension the cultures are sampled. The samples are tested for their florescence intensity at the end of the experiment.

2)Then, to confirm the validity of the recombinase system as a “bilateral” switch, we need to prove that it can flip the sequence between the recombination sites, in this case, the promoter, back and forth for rounds of periods. Following this goal, we put a constitutive promoter flanked by attB and attP in between genes of two different florescent proteins, resembling the two pathways, on the reporter plasmid. And the expressor reporter consists of a constantly expressed integrase and a arabinose-induced RDF, such that when cultured in an arabinose-free medium, the microbe acts as they were designed for non-digestion time, while then cultured in a medium with arabinose, it behaves as they would during digestion inside human intestine. According to our previous investigation in human practice, we learned that the period of time that chyme spend inside human digestive track is about 6 – 8 hours, including approximately 4 hours inside stomach. Under these bases, we decide a period of 2 hours for induction.

Similar to the previous experiment, the strains are cultured for 5 hr before induction with 5 μM arabinose. After two hours, the medium is changed back to an arabinose-free one through centrifugation and resuspension. Then after another three hours another round of arabinose is added, then removed after two hours…… The cycle is repeated for five times. Samples are taken every hour and tested for their kind and intensity of florescence.

Degradation tag


For the recombinase system to function properly, RDF must appear and disappear rapidly in order to regulate the activity of integrase. Therefore, it is as important to produce RDF as to degrade it. While the former can be readily achieved, provided there is an appropriate expression system, the latter, on the other hand, relies on functionality of a protein degradation system. In order not to introduce additional burden to the microbe, we make use of already-existing degradation machinery of E. coli. Between SsrA and RepA, two frequently-used peptide sequences that induce degradation of protein, we choose RepA, for its functioning on the N-terminal prevents loss of the tag through nonsense mutation, and for its better ability to perform this task shown in previous research.

Two recombinase system are constructed, as is done in bilateral switch, one with repA prior to RDF under control of arabinose-induced promoter and the other without. Both groups are tested following same protocol as bilateral switch, and the rates of sequence flipping are compared.

Digestion sensors


Several candidates with potential to finish the task are tested and their functions compared. GFP is used as indicator in experiments. For inhibitory promoters, strains are cultured for 5 hours in inducer-containing media before cells are centrifuged and media refreshed with inducer-free ones, then begins sampling. For activative promoters, strains are cultured in inducer-free media before addition of inducer and sampling. Florescence intensity of the samples are tested to give induction curves.

Induced suicide


This module aims to enable users of this microbe to terminate engineered bacteria inside their intestines whenever needed. This mechanism is designed in a way such that the inducer specifically induces the suicide of microbes that are modified. On one hand, it has to eliminate the engineered microbe with enough efficiency; on the other, it has to be harmless to the unmodified microbe. Consequently, our experiments for this module is carried out from the following two perspectives.

First, to prove that by inducing expression of translation inhibitor MazF, we can promptly eliminate the engineered bacteria, the cultured strains are exposed to suicide inducer arabinose. The living bacteria in the samples are counted by diluting and spreading on solid LB-ampicillin medium.

Then, to prove that MazF kills the cell without lysing it, we measured OD600 of each sample to give an overall number of intact bacteria, dead and alive.

Kill switch


Kill switch aims to prevent environmental contamination from the engineered bacteria through a cold-triggered toxin system. The experiment protocol is as follows.

The strains are cultured overnight at 37 ℃ before equally divided into two flasks. The two flasks are then cultured separately in 27 ℃ and 37 ℃. Samples are taken every 3 hours and living cells counted by spreading diluted samples on Petri dishes.