Team:DLUT China/Design

Navigation highlighting using waypoints.js

Design 1
Design 2


Selection of chassis organisms:

The choice of chassis organisms is very important to our entire experiment. It determines not only the feasibility of our experiment, but also the difficulty of our experiment. We chose the chassis organism as Nissle e. coli because its genetic manipulation is relatively simple and it is also a intestinal probiotics without damage to the host. The exocrine system of Nissle e. coli is relatively mature, and some breakthroughs have been made in recent years [1,2]. Therefore, Nissle e. coli is one of the most promising engineered strains currently used to deliver proteins to host cells. This is also why we choose Nissle e. coli.

The plasmid we use is pet-22b. We commissioned CPT to synthesize our gene fragment at the polyclonal site of pet-22b and used Amp resistance genes on the plasmid to screen our transformed strains. We initially used the BL21 escherichia coli strain to express our protein and used the dh5-e escherichia coli strain to store our plasmids.
Signal peptide selection:

In order to successfully secrete our uric acid oxidase outside the cells, we used 4 different signal peptides for secretion experiment, and selected the signal peptides with the best secretion effect to be added into the sequence. We used the signal peptides to deliver GFP and measured the fluorescence data of GFP to compare the effects of 4 different signal peptides.

We decided to use 4 signal peptides (OpmA, PeLB, phoA, STiI) for secretion test, and designed the following 5 sequences to test our secretion system:

1.Sac I-Uric acid oxidase-Sal I
2.Nde I-OpmA-Sac I-GFP-Sal I-TAT-Terminator-Hind III
3.Nde I-PeLB-Sac I-GFP-Sal I-Cyclic peptide-terminator -Hind III
4.Nde I-phoA-Sac I-GFP-Sal I-HIV Penetrating peptides-Hind III
5.Nde I-STiI-Sac I-GFP-Sal I-R8-Hind III
(The purple one is the restriction site.)
Design of uric acid response system

We used transcriptional regulator HucR[3] to sense uric acid concentration. Transcriptional regulator HucR is from d. radiodurans strain, belonging to MarR family of transcriptional regulator. This transcription factor binds a single binding site in the promoter region Shared by both genes through high affinity, inhibiting its own expression and the expression of adjacent uric acid enzyme [4]. The transcription regulator HucR binding site is called hucO. HucO is the common sequence of HucR and uric acid oxidase promoter in radioluccus resistant coccus. When uric acid was not present, HucR protein bound to hucO and blocked the binding of RNA polymerase to block the expression of related genes. In the presence of uric acid, the binding of HucR protein to hucO was inhibited by uric acid molecules, so that HucR protein could not interact with hucO, thereby activating the expression of related genes.

We also added Uric Acid Transporter (ygfU) after HucR sequences, making our sequences more sensitive to Uric Acid concentration.

Figure 1 We used HucR to control uric acid oxidase expression when uric acid concentration was lower than that of the system.At the threshold value, HucR inhibited the expression of the system. When the uric acid concentration was higher than the threshold value, HucR inhibition of the system was removed by uric acid molecules, and uric acid oxidase was normally expressed by the system.
Cell osmotic peptide selection:

We decided to use 4 kinds of cell osmotic peptides (TAT, cyclopeptides [5], HIV osmotic peptides, R8) to carry out the experiment of urea oxidase penetrating intestinal wall cells, and use caco-2 human colon cancer cell lines to carry out the experiment of cell osmotic peptides, and culture caco-2 cells on transwell membrane. After the cells have grown into a dense layer, DMEM culture medium containing urate oxidase with osmotic peptide is added to the membrane to determine the content of uric acid oxidase below the membrane, so as to judge the effect of different osmotic peptides.

Among the four selected cytoosmotic peptides, cyclopeptides were found in reference 5 to deliver phages from the intestines of mice to the blood of mice.
Microbial population control system:

In order to prevent the excessive growth of our Nissle escherichia coli in patients' intestines, disrupt the balance of intestinal flora in patients and affect the physical health of patients, we decided to set up the quantity control system of bacteria flora. We refer to the 2016 imperial flora control system, using lasR induced e. coli secretion of N - acetyl homoserine lactone, when the concentration of N - acetyl homoserine lactone after more than a threshold, Las lasR activates expression promoter downstream genes. We designed Gp2 in the downstream gene. Gp2 is an RNA polymerase inhibitory protein that inhibits the growth of escherichia coli, so as to control the quantity of Nissle escherichia coli in the intestinal tract.
Figure 2 We use lasR and Las promoter to control the expression of Gp2, when N - acetyl homoserine lactone concentrations below the threshold of the system, and PLas is in closed state, not express Gp2; When the concentration of n-acetyl hyperserine lactone was higher than the threshold value, lasR activated PLas, leaving PLas in an open state, and the system expressed Gp2 normally.
Lethal system:

In order to prevent the loss of our Nissle escherichia coli to the external environment, compete with the dominant local strains, and disrupt the normal ecological balance of the environment, we decided to set up in vitro lethal system. Since we were involved in more sequences, we decided to design two plasmids and introduce them into one bacterium at the same time. Therefore, we also set up the plasmids loss killing system.

We designed two systems.

System 1:
Figure 3 The Figure above is plasmid dlut1, mainly used to express uric acid oxidase; Here is plasmid dlut2, which is responsible for lethal systems.

SRNA in plasmid 1 is an inhibitory element of lysis2 in plasmid 2, which inhibits lysis2 expression. SRNA in plasmid 2 is an inhibiting element of lysis1 in plasmid 1, which inhibits lysis1 expression. When one of the plasmids is lost, the lytic protein of the other plasmids will not be inhibited, that is, the bacteria will express the lytic protein and cause the bacteria to lyse, thus killing the bacteria that lose the plasmids, so as to prevent the loss of plasmids.

Due to the high concentration of uric acid in the intestines of hyperuricemia patients, we use uric acid as the response signal of the lethal system. When the uric acid concentration exceeds the threshold, HucR is inhibited by uric acid, and Phuco is in the open state. Subsequent genes can be transcribed and translated normally, but since lysis1 is inhibited by sRNA, the bacteria will not be cracked. When the uric acid concentration was lower than the threshold value, HucR inhibited Phuco, and Phuco was turned off. Subsequent genes could not be transcribed and translated, so that sRNA inhibiting lysis2 could not be expressed, while lysis2 could be normally expressed, resulting in death of bacteria. Due to the high concentration of uric acid in the intestinal tract of uric acid patients, the death rate of bacteria in the patient is not high. However, when the bacteria are lost to the external environment, the uric acid concentration in the external environment is lower than the threshold value, which activates the lethal system of the bacteria, leading to the death of bacteria cracking and reaching the purpose and effect of in vitro death.

System 2:
Figure 4 Above is plasmid dlut3, mainly used to express uric acid oxidase; Here is plasmid dlut4, responsible for lethal systems.

SRNA in plasmid 1 is an inhibitory element of lysis2 in plasmid 2, which inhibits lysis2 expression. SRNA in plasmid 2 is an inhibiting element of lysis1 in plasmid 1, which inhibits lysis1 expression. In other words, the plasmid loss lethal mechanism of the system is the same as that of system 1.

Death in vitro was controlled by pCold to lysis3. PCold is cold shock plasmid in vitro in 16 ℃ under the condition of using IPTG induction of 3 h can activate lysis3, killed bacteria cracking, achieve the purpose of in vitro to death and effect. PCold came from the IGEM team of Northeastern University, and our two teams had a friendly interlab exchange.

[1] Powale, Urmila. 2016. Engineering Probiotic E. Coli With a Type III Secretion System for Targeted Delivery of Therapeutic VHH. Master's thesis, Harvard Medical School, 2016.
[2] IY Hwang, E Koh, A Wong, et al. Engineered probiotic Escherichia coli can eliminate and prevent Pseudomonas aeruginosa gut infection in animal models[J]. Nature Communications, 2017.
[3] T Bordelon, SP Wilkinson, A Grove, et al. The crystal structure of the transcriptional regulator HucR from Deinococcus radiodurans reveals a repressor preconfigured for DNA binding[J]. Journal of Molecular Biology 2006, 360(1):168-77.
[4] C Liang, D Xiong, Y Zhang, et al. Development of a novel uric-acid-responsive regulatory system in Escherichia coli[J]. Applied Microbiology and Biotechnology, 2015, 99(5): 2267–75.
[5] S Yamaguchi, S Ito, M Kurogi-Hirayama, et al. Identification of cyclic peptides for facilitation of transcellular transport of phages across intestinal epithelium in vitro and in vivo[J]. J Control Release, 2017, 28(262): 232-238.