Overview

The results of all our design will be shown in this page. So far we had finished parts of our scheduled experiments and the results had proved the reliability and feasibility of our design to some extent. However, some picture may be not that beautiful because of the lack of time, and we are still conducting experiments for a better results. Besides, the results of verification of Δ9-THC-sensing system and the activity of the enzymes will soon be added!!!

THC-sensing module

C-Phycocyanin extracellular secretion under IPTG induction

Verification of Plasmid Construction

We inserted the gene fragment into the plasmid PET-Duet1 in two steps by restriction endonuclease cleavage and ligation. Then we verified the correctness of plasmid construction and product length by colony polymerase chain reaction and gel electrophoresis. The gel electrophoresis pattern is as follows:

Fig.1 Gel electrophoresis results of the colony PCR of the plasmid Lane M: Marker III; Lane 1-2: colony PCR products of the T7-C-Phycocyanin plasmid(1237bp) Fig. 2 pET-Duet1- pInteraction-IPTG sequencing map

C-Phycocyanin Purification Verification

We transformed E.coli BL21(DE3) with our validated plasmid and carried out the function verification experiments as we described in the Experiment section. At first, we attempted to take pictures of secreted C-Phycocyanin with the optical microscope, however, the images didn’t show much difference between test group and control group and couldn’t show the blue. We guessed that the amount of protein exocytosis was too small to be seen by the optical microscope and we chose to use SDS-PAGE gel to verify the presence of protein.

Fig.3 SDS-PAGE map of C-Phycocyanin alpha subunit secreted extracellularly by E.coli Lane M1&Lane M2: Premixed Protein Marker(Broad); Lane 1-2: IPTG induced positive transformant; Lane 3-4: IPTG induced positive transformant passing through his-tag protein extraction kit. (C-Phycocyanin alpha subunit is approximately 20.6kD)

C-Phycocyanin extracellular secretion under Δ9-THC induction

C-Phycocyanin Purification Verification

Δ9-THC is banned for private sale in China, we chose the non-psychoactive 11-OH-Δ9-THC to test the normal function of the sensing protein. However, the 11-OH-Δ9-THC is soluble in methanol for transport, it was not until recently that we isolated 11-OH-Δ9-THC from methanol. We have planned Δ9-THC induction experiments (see our experiment) and are working hard to carry out these experiments. If there are subsequent experimental results, we will update our wiki!

ΔTHC metabolism module

Verification of Δ9-THC Metabolism Pathway and Parts

We construct the Δ9-THC metabolism pathway of cyp2c9, cyp2c19 and ugt1a3 which induced by Δ9-THC.

Verification of plasmids Construction

We insert the gene fragments of cyp2c9/cyp2c19/ugt1a3, ompT and His-tag into the pET-Duet1 downstream of the t7 promoter and upstream of the t7 terminator, and verify the bacterial colony PCR products by gel electrophoresis to initially determine the correctness of the plasmid construction. The results of gel electrophoresis bands of the plasmids are shown in Fig.4- 6:

Fig. 4 Gel electrophoresis results of cyp2c9 (100% positive rate) Fig. 5 Gel electrophoresis results of cyp2c19 (87.5% positive rate) Fig. 6 Gel electrophoresis results of ugt1a3 colony PCR (100% positive rate)

After that we extract the plasmids and verify the correct construction of the plasmids by sequencing them by the company. The plasmid profiles returned by the company are shown in Fig.7-9.

Fig.7 pET-Duet1- cyp2c9 sequencing map Fig.8 pET-Duet1- cyp2c19 sequencing map Fig.9 pET-Duet1-ugt1a3 sequencing map

Protein Expression, Purification Verification

The fusion expression proteins are purified by His-Tag protein extraction kit and verified by SDS-PAGE gel. The results of the gradient elution SDS-PAGE of the three target proteins are shown in Figures 6-8. The results show that the proteins are successfully expressed.

Fig.10 The gradient elution SDS-PAGE results of CYP2C9 (the target protein bands are in the red box) Fig.11 SDS-PAGE results of gradient elution of CYP2C19 (the target protein bands are in the red box) Fig.12 SDS-PAGE results of gradient elution of UGT1A3 (target protein bands in red box)

Suicide Module

We use two safety modules to keep the engineered bacteria under restriction. First, arabinose suicide induction module allows users to terminate the product at any time. Second, the use of 37℃ temperature control suicide switch prevents engineering bacteria strain from polluting the environment outside human body.

Verification of Plasmid Constructione

To verify the RNA thermometer, we replaced mazE and mazF following temperature controlled RBS fragment with GFP on the plasmid.

Bacterial colonies PCR products were verified to be correct by gel electrophoresis. The gel electrophoresis bands of the plasmid were as Fig.13:

Fig.13 Gel electrophoresis results of colony PCR (Lane10:RNA Thermometer Verification,5119bp)

After that we extract the plasmids and verify the correct construction of the plasmids by sequencing them by the company. The plasmid profiles returned by the company are shown in Fig.14.

Fig.14 Results of plasmid RNA Thermometer Verification sequencing

Conduct confirmatory experiments

Arabinose-induced kill switch

L-arabinose is commonly used to induce the engineered bacteria to stop proliferation. It is also a way to prevent engineered bacteria from spreading and leaking into the soil, because there is enough arabinose in the soil. We used five different concentrations of arabinose (0%, 0.05%, 0.1%, 0.15%, 0.2%) in the experiment to induce the engineered bacteria. Samples were taken every hour to measure the OD600 to get the total number of unruptured bacteria. The killing effect was observed by counting the viable cells by spreading diluted samples on plates. The data of OD600 measurement are shown in Fig.15 and the growth of colonies is shown in Fig.16.

Fig.15 The growth of engineered bacteria in different arabinose concentration Fig.16 Comparison of growth of the engineered bacteria treated with L-arabinose at different concentrations and time duration. (from left to the right: 1h, 2h, 3h, 4h; top to bottom: 0%, 0.05%, 0.1%, 0.15%, 0.2%) Fig.17 The numbers of colonies after incubating in different arabinose concentrations

Through spreading plate verification(Fig.18) combined with modeling results(Fig.19), most of the engineered bacteria die at 2749s which means that the system has the high killing efficiency. And further calculation showed that the optimum killing concentration is 0.1%.

Fig.18 Comparison of growth of the engineered bacteria treated with 0.1% L-arabinose at different time duration, the dilution of the bacterial solution is also different. (A) 0.5h, 10⁶ (B) 0.75h, 10⁵ (C) 1h,10⁵ (D) 1.5h, 10⁵ (E) 2h, 10⁵ (F) 2.5h, 10⁵ (G) 3h,10⁵ (H) 3.5h,10⁴ (I) 4h, 10^3 Fig.19 The numbers of remaining live cells incubated in 0.1% arabinose LB medium over time.

Cold-triggered toxin suicide switch

In order to prevent the engineered bacteria from polluting the environment outside human body, the 37℃ temperature control suicide switch was introduced, and the toxin-antitoxin system was used to realize the suicide of engineered bacteria in the non-37 ℃ environment. The strain was incubated at 37 ℃ and then divided equally into two conical flasks. Two conical flasks were incubated at 28℃ and 37℃ for shock culture. Samples were taken hourly for OD600 measurements to get the total number of unruptured bacteria, and the killing effect was observed by counting viable cells by spreading diluted samples on plates. The data of OD600 measurement are shown in Fig.20 and the expression of mazE is shown in Fig.21.

Fig.20 The data of OD600 measurement. Fig.21 The expression of mazE, mazF and mazEF at different temperatures and different incubation time.

On the left side is the situation where temperature ≥ 37 ℃, and concentration of mazF, the toxin, remains at single digit throughout the 2-hour simulation, which is way lower than the cleavage threshold. While the simulation result of ≤37 ℃ on the right side implies that mazF can be produced at a pretty fast rate, its degradation rate is slow enough to be ignored, and there is not much mazE that binds to it. Thus, it can eventually hold a high concentration to be toxin to cells. In this case, the concentration of the antitoxin mazE peaks around the first few tens of seconds and then declines slowly until 2 hours later. Hence it's safe to draw the conclusion that the bacteria can be eliminated for some degree when leakage happens. We will further explain how we developed the suicide module and explore the probable principle for low killing efficiency.

Reflection：We constructed a toxin-antitoxin system controlled by RBS thermometer, and the experimental results showed that the killing effect of RBS thermometer on engineered bacteria by inhibiting mazE expression at 28℃ did not reach our expectation. We obtained two hypotheses by analyzing the experimental results and literature.

1. The concentration gap between MazE and MazF caused by RBS inhibition of MazE at 28℃ was not enough to kill the engineered bacteria quickly. Moreover, the engineered bacteria had negative feedback regulation of MazEF in their chromosomes, and the up-regulation of maze expression by the negative feedback regulation mechanism made the engineered bacteria free from MazF killing after several hours of culture.

2. According to some literatures, the effect of MazF is growth arrest rather than programmed cell death. Therefore, it is possible that in the case of dormancy of engineered bacteria, we placed them at 37 ℃ after coating, mazE expression was restored, and engineered bacteria were released from growth inhibition.

We wanted to verify the second hypothesis, so the engineered bacteria were over-diluted and coated on LB-Kanr solid medium and cultured at 28 ℃, and then a petri dish was taken every hour and placed at 37 ℃ for a total of 8 hours to see if single colony can recover at better circumstances. Colonies were then incubated for 5 hours to be seen. As the outcome shows, all two colonies treated for less than 7 hours recovered compared to the control group grown in 28℃. And those treated for more than 6 hours suffer from proliferation inhibition to different degrees. However, low concentration leads to high possibility of coincidences, further experiments of higher concentration will be conducted later and results of our current exploration are shown below in Fig. 22.

Fig.22 Comparison of growth of the engineered bacteria for different time cultured at 28℃( (A) 5h (B) 6h (C) 7h (D) the control group) and then recovered at 37℃, the initial dilution of the bacterial solution is the same .

RNA Thermometer Verification Pathway

To verify the function of RNA thermometers, strains were cultured at 37 °C and 28 °C respectively, and the initial OD600 and GFP fluorescence intensity were measured. Then samples were taken every half an hour to measure the OD600 and GFP fluorescence intensity.

GFP measurement results are shown in Fig.23. And we used a fluorescence microscope for observation. The bacterial liquid under the fluorescence microscope is as Figure 24:

Fig.23 GFP fluorescence intensity after incubating in different temperatures. Fig.24 Images under fluorescene microscope. (A) No GFP in engineerd bacteria incubated at 28℃. (B) Bright field image of bacteria incubated at 28℃. (C) The field of view after superposition of A and B. (D) GFP in engineerd bacteria incubated at 37℃. (E) Bright field image of bacteria incubated at 37℃. (F) The field of view after superposition of D and E. (fluorescene microscope 10 x 100).