index

Demonstration of the smell sensing module

Demonstration of the reproduction module

Demonstrate

Following our design, we have carried out a series of experiments carefully and considerately. We've worked out lots of results which make us delighted. We've verified that our smell sensing module, memory module and erasure module can work as expected. Though we've encountered a lot of problems while verifying the reproduction module, we also have tried various approaches to reach our goal.

Here we are going to demonstrate what we've successfully done and what are under verification.

. Demonstration of the smell-sensing module

To enable the engineered bacteria to sense benzyl alcohol, we use the P_are benzyl alcohol inducible promoter from Acinetobacter sp. Strain ADP1 as the core component of this module. P_are is an AreR-regulated σ⁵⁴-dependent promoter in Acinetobacter sp. Strain ADP1 and can be induced by benzyl alcohol. The regulatory protein of P_are is AreR, a member of the NtrC/XylR regulatory protein family. This type of enhancer protein can bind to the repeat region upstream of the P_are promoter after activated by benzyl alcohol or other aromatic compounds. In the case of cofactor-induced DNA looping and activated AreR presence, a closed promoter complex will be formed with RNA polymerase to turn on the transcription of downstream genes [1].

In this module, we express AreR with J23102 constitutive promoter, and use P_are to regulate the expression of LuxI. LuxI can synthesize AHL, which can further activate the downstream signaling pathway, for example, the memory module in our project (Figure 1). To verify the function of this module, we replaced luxI with gfp and induced its expression with different concentrations of benzyl alcohol (Figure 2). A significant difference in fluorescence intensity was observed in experiment (Figure 3).

Figure 1. Design of the smell-sensing module.

Figure 2. Verification of the smell-sensing module.

Figure 3. Induced GFP by benzyl alcohol. A. Kinetic curves of fluorescence intensity induced by different concentrations of benzyl alcohol. B. Fluorescence intensity induced by benzyl alcohol of different concentrations for 8h. The experiments are carried out in three biological replications. The error bars indicate standard error.

As can be observed from the above charts (Figure 3), when the induction time was up to eight hours, the highest fluorescence intensity was observed in the experimental group with 3.8 mM benzyl alcohol, with a difference of 4~5 times larger than the control group without induction. Therefore, we think that this module can work. It can express luxI gene after sensing benzyl alcohol, thus transmitting the signal to the memory module.

. Demonstration of the memory module

o Demonstration of taRNA-crRNA interaction

To ensure the basic elements in our AND gate can work, we designed a circuit to verify the function of taRNA-crRNA pair (Figure 4). As we've explained in the Design page (https://2019.igem.org/Team:HZAU-China/Design), crRNA can form a hairpin structure that can block the RBS sequence, preventing the combination of ribosomes to RBS, which can inhibit translation.

Figure 4. taRNA-crRNA interaction detected by sfGFP.

We verified the function of taRNA-crRNA pair by adding IPTG into the medium. IPTG would induce promoter P_A1lacO-1 to produce taRNA. taRNA would interact with crRNA driven by the constitutive promoter J23102 and unleash the inhibition of crRNA which will activate the expression of sfGFP [2]. Comparing the intensity of sfGFP of the groups with IPTG supplementation and the group without IPTG supplementation will verify the function of this circuit.

Figure 5. taRNA-crRNA interaction detected by sfGFP. OD₆₀₀ was diluted to 0.3, and then the bacteria were incubated for 4 hours at 37℃ 200rpm before 1mM IPTG was added into the culture media. After induced for another 4 hours, the fluorescence intensity (Excitation: 485nm, Emission: 528nm) and OD₆₀₀ were measured by a microplate reader (Synergy H1 hybrid multi-mode reader). Significant fold-change could be observed, indicating that the taRNA-crRNA circuit was functional. The experiments were carried out in three biological replications. The error bars indicate standard error.

From Figure 5, we can observe that after adding IPTG into the medium, significant fold-change appears between groups with IPTG supplementation and groups without IPTG supplementation. The result proves that this circuit can work. taRNA do unleash the inhibition driven by crRNA and activate the translation of downstream genes.

o Demonstration of the switch

To identify whether the AND gate can play as a switch in our project, we designed the circuit shown in Figure 6. The design of this circuit was advised by the AND Gate Model described in the model page, which suggested us take taRNA as the accumulated substances rather than crRNA.

At first, AHL was added to induce the expression of LuxI-LVA and taRNA. Due to the positive feedback, taRNA would accumulate inside bacterial cells. After inducing for 8 hours, 1 mM IPTG was added to activate the transcription of crRNA and sfgfp on its downstream. If taRNA could 'unlock' crRNA, we could detect green fluorescence after adding IPTG.

Figure 6. Verification of the switch circuits.

Figure 7. Verification experiment of the switch. At first, 10nM AHL was added into both the experimental (+IPTG) and the control (-IPTG) group to accumulate taRNA. After inducing for 8 hours, 1mM IPTG was added to the experimental group (+IPTG) in order to transcribe crRNA and sfgfp. Fluorescence intensity (Excitation: 485nm, Emission: 528nm) and OD₆₀₀ were measured by a microplate reader (Synergy H1 hybrid multi-mode reader). The result shows that the fluorescence intensity of the experimental group is significantly higher than the control group, indicating that the switch can function properly. The experiment was carried out in three biological replications. The error bars indicates standard error.

From Figure 7 we can see that if memory module has accumulated enough amount of taRNA, the addition of IPTG can unlock the translation of downstream genes. The fluorescence intensity of the experimental group (+IPTG) is significantly larger than the control group (-IPTG), indicating that the switch can work. In this way, we can switch on the reproduction module by human's decision.

o Demonstration of the leakage control

We’ve tried 3 plans to decrease the leakage of the positive feedback circuit as described in the Design page (https://2019.igem.org/Team:HZAU-China/Design). According to the Control Leakage Model described in the model page, Plan I and II are inappropriate to gain the leakage control effect. The inability of the Plan I and II was also proved in our experiment, indicating that the model is effective.

Plan III has reached the best effect according to our test. The mutation of lux box of luxPR promoter has effectively changed the binding characteristics of LuxR protein to lux box, which significantly decreases the leakage of the positive feedback circuit [3]. The fourth and the twelfth bases of the original luxPR were mutated to G and T simultaneously. We named this mutant as luxPR-4G12T and submitted it as our improved part this year. You can find more information about this part here: http://parts.igem.org/Part:BBa_K3205005.

Figure 8. The standard fluorescence by AHL of different concentrations after 8 hours of incubation. Fluorescence intensity and OD values were measured by microplate reader respectively at 528 nm and 600 nm wavelength. The experiment was carried out in three biological replications. The error bars indicate standard error.

It was observed that the expression intensity of sfGFP of the mutant was low at a low concentration of AHL, and the intensity remained stable. But the wild type had a certain degree of leakage without AHL. And we discovered that when the concentration of AHL reached 10^-1nM, the fluorescence intensity of wild type had increased, but the mutant didn't change much. When the concentration went up to 10²nM, the fluorescence intensity of the mutant and the wild type became similar. In contrast, the mutant had a very low leakage, high threshold, and unchanged intensity (Figure 8).

Compared to the wild type, the mutant was more suitable for our project. The biggest problem of our project was that the memory module would start itself due to the leakage of the promoter. Since the leakage of the mutant was very low and the threshold to activate this promoter was high, the mutant needed more time and a higher concentration of inducer to active the memory module than the wild type.

o Demonstration of the positive feedback

Due to the instability of RNA in vivo, if we want to keep the concentration of taRNA in vivo, we can express it constantly to offset its degradation. This experiment constructed two circuits (Figure 9) to demonstrate whether the positive feedback could work. At first, we used same concentration of AHL to induce the expression of downstream genes. If worked, AHL will be produced and accumulated in the bacterial cells under synthesis of LuxI, but the bacterial cells without luxI gene won't [3]. Theoretically, if dilute the culture medium with same dilution factor, the fluorescence intensity value of the group which do not contain luxI gene won't arise or stay low, but the fluorescence intensity value of group containing luxI gene will arise and will be higher than the former. AHL produced by LuxI protein will accumulate in bacterial cell despite dilution, so it can still activate the expression of mRFP1 and LuxI. But the other one without luxI gene won't arise, since AHL's concentration is too low to activate the expression of downstream genes.

Figure 9. Verification circuit of the positive feedback.

Figure 10. Verification experiment of the positive feedback module. At first, 10nM AHL was added to induce both the experimental (+luxI) and the control (-luxI) group to induce mRFP1. Eight hours later, we diluted the bacteria to OD₆₀₀ = 0.1 with the same dilution factor, and then detect OD₆₀₀ value and fluorescence intensity value (Excitation: 581nm, Emission: 607nm) every 20 minutes for another 8 hours. The result shows that the fluorescence intensity of the experimental group is significantly higher than the control group, indicating that the positive feedback module can work. The experiment was carried out in three biological replications. The error bars indicate standard error.

From Figure 10 we can figure out that after adding 100ng/mL ATc (anhydrotetracycline) into the medium, the fluorescence of the experimental group (+luxI) arose significantly. In contrast, the fluorescence of the control group (-luxI) hardly varied. In terms of these results, we state that the positive feedback does exist and can work well, which proves that our design of memory is successful.

. Demonstration of the reproduction module

The reproduction module is the most challenging module in our project for it involves in metabolic engineering. Metabolic engineering is a systematic engineering, because each substrate may involve in many reactions. Due to this, the optimization of the metabolic fluxes is important and challenging.

Taking this into consideration, we used E. coli W3110 BCAE as our chassis (W3110 ΔtyrB::FRT, ΔaspC::FRT, tyrA16::Tn10, ΔtrpE::FRT) within pSUFAAQ (pSU2718 derivative, aroF^fbr, pheA^fbr, lacI^q, hmaS_Ao) strain which is generously provided by Zhoutong Sun and Sheng Yang from CAS Center for Excellence in Molecular Plant Sciences / Institute of Plant Physiology and Ecology [4].To make the bacteria grow normally, at least three amino acids should be supplemented into the culture medium, including L-tyrosine, L-tryptophan and L-aspartic acid. The supplementation of these amino acids was advised by the Metabolic Model described in the model page.

We successfully constructed a plasmid containing genes vcm17, mdlB and mdlC which are regulated by trc promoter. We then transformed this plasmid into the chassis strain. The three enzymes along with endogenous alcohol dehydrogenases can catalyze phenylpyruvate to benzyl alcohol theoretically [5]. However, we didn't detect the presence of benzyl alcohol by running HPLC after inducing the bacteria for 12-24 hours.

At first, we doubted that the genes of the enzymes hadn't been transcribed. To find out whether these genes were transcribed, we extracted whole RNA of the bacteria and carried out RT-PCR. The cDNA obtained from RT-PCR were then PCR-amplified by specific primers in order to detect the presence of the mRNA transcribed from gene vcm17, mdlB and mdlC (Figure 11).

Figure 11. RT-PCR results of the benzyl alcohol synthesis circuit. From left to right: line 1 is marker; mdlB+, mdlC+ and vcm17+ are the results of gene mdlB, mdlC and vcm17 after the bacteria were induced with 0.01mM IPTG for 3 and half hours; mdlB-, mdlC- and vcm17- are the results of gene mdlB, mdlC and vcm17 without the induction of IPTG under the same condition; mdlB', mdlC' and vcm17' are blank controls (without templates in PCR ). The results show that whether induced or not, all three genes have been transcribed.

Since the three genes are transcribed, we then transfer our focus to the detection of the proteins coded by these three genes. We plan to attach 6×His tag to each of the protein in the plasmid respectively, and extract the proteins by affinity chromatography. Then, we are going to detect the proteins by SDS-PAGE electrophoresis. If target proteins of right weight can be found on the gel, the translation of the proteins can be proved.

Besides the detection of the proteins of interest, we also need to know whether these enzymes can play their roles. Substrates feeding experiments will be used to find out whether these enzymes can work. After adding phenylpyruvate, phenyl glyoxylate and benzaldehyde, products will be detected by HPLC. Then, we will analyze the results and find out which enzyme doesn't work properly.

Conducting the works described above, we expect to find which part of the circuit can't work properly. We aim to complete these works before the presentation in Boston.

. Demonstration of the erasure module

In order to ensure the positive feedback can be terminated, we constructed a circuit (Figure 12) to demonstrate. At first, the positive feedback will be activated by AHL, and after that, more and more AHL and mRFP1 will accumulate in the engineered bacterial cells [6]. After inducing for 8 hours by AHL, ATc (anhydrotetracycline) was added into the media, and the OD₆₀₀ value and the fluorescence intensity were detected every 20 minutes for 8 hours. If the erasure module works, we can see the fluorescence intensity of mRPF1 will be much lower than that of the one without ATc, due to the hydrolyzation of AHL.

Figure 12. Verification circuit of the erasure module.

Figure 13. Verification experiment of the erasure module. At first, 10nM AHL was added into both the experimental (+ATc) and the control (-ATc) group to induce mRFP1. After inducing for 8 hours, 100ng/mL ATc was added to the experimental group (+ATc) to express AiiO. Fluorescence intensity (Excitation: 581nm, Emission: 607nm) and OD₆₀₀ were measured by a microplate reader (Synergy H1 hybrid multi-mode reader). The result shows that the fluorescence intensity of the experimental group is significantly higher than the control group, indicating that the erasure module can work. The experiment was carried out in three biological replications. The error bars indicate standard error.

Obviously, after adding 100ng/mL ATc, enzyme AiiO of the erasure module began to express and work, starting to limit the accumulation of AHL [7]. Comparing with the control group (-ATc), the fluorescence intensity of the experiment group (+ATc) was about 13 times lower (Figure 13). The curve of the fluorescence intensity/OD₆₀₀ of the group in which ATc was added was nearly horizontal (Figure 13), which meant that AHL generated by LuxI were hydrolyzed by AiiO. To sum up, the function of the erasure module has been proved.

. Demonstration of our hardware

In order to make our biological machine easier to use and more human-friendly, we built hardware for our project. The hardware consists of 3 layers. The first layer is a big warehouse that stores fresh culture medium and inducers (IPTG, ATc and the smell molecules we want to memorize). The second layer is an incubation chamber for bacteria incubation. Tubes are used to connect the first layer and the second layer for the addition of the inducer. Cameras are set on the second layer to inspect the reporter signals generated by the bacterial cells. Blenders are set inside the chamber for bacteria incubation. The third layer is essentially a waste box used for collecting waste fluids from the second layer, avoiding polluting the environment.

We also designed software to control the hardware with mobile phones. The software makes the hardware an extension of mobile phones. Pressing the button "Start Recording", the smell molecules will be added into the second layer. Pressing "View Status", the camera on the second layer will check the status of the bacterial cells, reporting whether they've memorized or not. Pressing the "Reproduce" button, IPTG from the first layer will be added into the second layer, initiating the reproduction module. Pressing the "Erase" button, ATc from the first layer will be added into the second layer, resulting in memory erasure.

For more details about our software, please visit: https://2019.igem.org/Team:HZAU-China/Software

For more details about our hardware, please visit: https://2019.igem.org/Team:HZAU-China/Hardware

Reference

[1] Jones R M, Williams P A. areCBA Is an Operon in Acinetobacter sp. strain ADP1 and Is Controlled by AreR, a ς54-Dependent Regulator[J]. Journal of bacteriology, 2001, 183(1): 405-409.

[2] Isaacs F J, Dwyer D J, Ding C, et al. Engineered riboregulators enable post-transcriptional control of gene expression[J]. Nature biotechnology, 2004, 22(7): 841.

[3] Egland K A, Greenberg E P. Quorum sensing in Vibrio fischeri: elements of the luxI promoter[J]. Molecular microbiology, 1999, 31(4): 1197-1204.

[4] Sun Z, Ning Y, Liu L, et al. Metabolic engineering of the L-phenylalanine pathway in Escherichia coli for the production of S-or R-mandelic acid[J]. Microbial cell factories, 2011, 10(1): 71.

[5] Pugh S, McKenna R, Halloum I, et al. Engineering Escherichia coli for renewable benzyl alcohol production[J]. Metabolic Engineering Communications, 2015, 2: 39-45.

[6] Pai A, Tanouchi Y, You L. Optimality and robustness in quorum sensing (QS)-mediated regulation of a costly public good enzyme[J]. Proceedings of the National Academy of Sciences, 2012, 109(48): 19810-19815.

[7] Li Y, Zhao J, Quan C, et al. Production, characterization, and powder preparation of quorum quenching acylase AiiO for pathogen control[J]. Preparative Biochemistry and Biotechnology, 2019, 49(2): 151-157.

Team:HZAU-China/Demonstrate