Assisted Living Home Care

The first iteration

Design

Chronic lower gastrointestinal bleeding is a common clinical symptom, but the traditional detection method of intestinal micro bleeding is more complex and may bring bad experience to patients. Therefore, we reviewed the relevant literature and established a pathway for heme transport and visual expression containing four genes in E.coli, that is, ChuA protein can transfer the extracellular heme to the cell, and the intracellular heme further combines with the heme binding protein HrtR, changing the HrtR conformation, leading to the activation of the promoter HrtO, and then it will induce the expression of the downstream reporter gene cjBlue, producing blue pigment in bacteria.

 

Build

Plasmid construction

Because our laboratory has an off-the-shelf source of pET28a (+) vector and we often use this vector and are most familiar with it, so we decided to use it as the vector to verify the feasibility of our design, so we constructed pET28a (+)-ChuA-HrtR-HrtO-cjBlue and transformed it into E. coli BL21 (DE3).

 

Figure 1 The plasmid map of pET28a (+)-ChuA-HrtR-HrtO-cjBlue

 

Strain establishment

We u se d   E.coli DH5α to colon e the  plasmid and then transformed it into E.coli BL21(DE3) to establish the expression strain.

 

Learn

After  experimental test ing , we found that the expression of pigment was extremely low or even no. By  review ing the literature and consulting our PI and advisor, we learned that the reason for this phenomenon m ight  be that the four genes used in this study were linked to a plasmid vector, so that the plasmid was too large and the product expression was unstable. Therefore, we decided to divide the target gene into two parts and introduce them into two plasmids, and then transform them into one s train at the same time.

 

The s econd iteration

Design

We use d  pET28a(+) vector to synthesize the pET28a(+)-ChuA - HrtR plasmid, and use d   the pUC19 - HrtO and the pSB1C3-cjBlue plasmid in iGEM 2019 DNA Distribution Kit Plate 5（ http://parts.igem.org/Part:BBa_K864404 ） . We used PCR amplification and enzyme digestion to link the second plasmid pSB1C3-HrtO - cjBlue we needed.

 

Build

Plasmid construction

We constructed two recombinant plasmids : pET28a(+)-ChuA-HrtR and pSB1C3-HrtO-cjBlue .

 

 

Figure 2 Plasmid Maps of pET28a (+) - ChuA HrtR and pSB1C3-HrtO-cjBlue

 

Strain establishment

We used E.coli DH5α to colone the plasmid, and then we transformed two plasmids into E.coli BL21 to establish an expression strain.

Figure 3 Transformed colony on the LB culture medium

 

Test

Escherichia coli transformation experiment

To confirm the successful construction of the target plasmid pSB1C3-HrtO-cjBlue, we first transformed the enzyme linked product into E.coli and coated it on the LB culture medium containing chloramphenicol antibiotic.

 

Figure 4 Enzyme linked bacterial colony on LB culture  medium

 

Agarose gel electrophoresis

Further verify whether the plasmid was successfully constructed.

 

Learning

In the second iteration, although we successfully constructed a strain containing pSB1C3-HrtO-cjBlue plasmid, we found mutation in the plasmid sequence after sequencing verification. Therefore, we would use In-Fusion technology in the third iteration to reduce non-specific amplification during enzyme digestion and achieved more efficient connection.

 

The t hird iteration

Design

We used an efficient assembly method——In-Fusion technology to insert HrtO and cjBlue sequences into a plasmid to confirm the successful construction of the target plasmid pSB1C3- HrtO-cjBlue.

 

Due to Covid-19, we could not borrow the instruments needed for electric transformation in time, so we decided to transform it by ordinary chemical transformation methods.

 

Test

E.coli transformation experiment

In order to confirm the successful construction of the target plasmid pSB1C3-HrtO-cjBlue, we used seamless cloning technology to insert HrtO and cjBlue sequences into a plasmid, and coated it on the LB medium containing chloramphenicol antibiotic. E. coli can only grow normally if it is successfully transferred into the plasmid.

Figure 5 The pSB1C3-HrtO-cjBlue plasmids which were successfully constructed by In-Fusion technology on LB culture medium

 

Finally, two plasmids PET28A (+)-ChuA-HrtR and pSB1C3-HrtO-cjBlue were successfully transferred into E. coli, but due to time constraints, we only demonstrated that heme was indeed able to activate cjBlue expression, so we didn't explore the minimum heme concentration.

 

Agarose gel electrophoresis

Further verify whether the plasmid was successfully constructed.

 

Figure 6 The running gel of pSB1C3-HrtO-cjBlue plasmid successfully constructed by In-Fusion technology

 

Figure 7 Transformed colony on LB culture medium

 

Learn

Because the strains we built are excreted through feces, and feces cannot be completely collected and sterilized, which will have some unpredictable effects. So we decided to add suicide switches inspired by last year's project.

 

The fourth iteration

Design

In terms of the design of the suicide switch, we decided to use the suicide switch that our team had verified last year to reduce the leakage problem and improve the safety of the sensor. Namely, nMag and pMag are inserted respectively in different domains of Cas9. They will form a dimer activated by blue light to form a complete Cas9, and then cut the target gene segment under the guidance of gRNA.

 

Figure 8 Blue light activated suicide switch

 

Test

The suicide switch was successfully verified last year (please click https://2021.igem.org/Team:NWU-CHINA-A/Implementation )

 

Learn

Because our sensor needs to enter the human intestinal tract to play a role, we chose E.coli Nissle 1917 as the expression strain instead of E.coli BL21. Since there was no T7RNA polymerase in E.coli Nissle 1917, we planned to use J23119 promoter to replace T7 promoter.

 

And considering that our suicide switch needs blue light activation,which is not available in normal homes. By looking through the literature we found another simple suicide switch , that is, to knock out a specific gene in E.coli.

 

The fifth iteration

Design

We imagine replacing the T7 promoter in the plasmid with the J23119 promoter, and since E.coli Nissle 1917 has been proved to be a probiotic that is safe and harmless to human beings, we wanted to replace the expression strain from E.coli BL21 to E.coli Nissle 1917.

 

We found another suicide switch with higher feasibility, that is, to knock out the dapA gene fragment in the E.coli chromosome. E.coli can only conduct cell wall biosynthesis and growth when exogenous diaminopimelate (DAP) are added. It is reported that the concentration of DAP in soil is insufficient to support the growth of cell wall, so as to achieve biological protection in vivo and environment.

 

Build

Plasmid construction

In the first constructed plasmid pET28a(+)-ChuA-HrtR, we updated the T7 promoter to the J23119 promoter, and planned to knock out the dapA gene fragment.

 

Strain construction

We wanted to transform two plasmids into E.coli Nissle 1917 through electrotransformation to establish an expression strain.

 

Test

E.coli transformation experiment

To confirm the successful construction of the target plasmid J23119-ChuA-HrtR, we transformed the enzyme linked product into E. coli and coated it on the LB medium.

 

Figure 9 Transformed colony on LB culture medium

However, due to Covid-19, our experiment time has been shortened, so the verification of the introduction of E.coli Nissle 1917 and the design of suicide switch have not been completed. In the next stage, we will verify it in the laboratory.

 

Engineering in model

Design: Our cooperation with DKU_China for hardware testing began with a short meeting. During the meeting, we introduced our needs and Shiran Yuan from DKU_China introduced some basic knowledge and concepts of modeling to NWU-CHINA-A to facilitate communication. Eventually, the two teams jointly selected the parameters for testing and completed the experimental design.

Test: For experimental results and quantitative analysis, please refer to our modeling page.

Learn: We analyzed the results of the first experiment together. The experimental results were enough to indicate that, despite the effects being slightly worse compared to conventional methods, the hardware works well. However, the data indicated that the error is relatively large. Eventually, we concluded that:

1、During calibration, the concentration of strains in the liquid used for calibrating was later found to be inconsistent, therefore indicating that the experimentally tested OD might deviate from the actual values.

2、Due to the problem that some of the team members in charge of operating the experiment lack experience, the operational errors in the experimental results are large.

Design: Learning from the previous cycle, the experimental design was adjusted. The calibration process was standardized, and the operation process was carried out by only one person to minimize operational errors.

Build: The basic experimental design stayed the same.

Test: For experimental results and quantitative analysis, please refer to our modeling page.

Learn: This time, from modeling we learned two useful results which indicate the success of our experiment:

1、The growth curve of the strains fits perfectly with the logistic growth model (theoretical prediction).

2、The hardware works well, and its difference in performance from the conventional hardware is statistically insignificant.

Quote

[1]  Tan ZiZhu,Li XiaoDan,Kong ChaoDi,Sha Na,Hou YaNan,Zhao KaiHong. Engineering Bacteria to Monitor the Bleeding of Animals Using Far-Red Fluorescence.[J]. ACS sensors,2021.

[2]  Isabella Vincent M,Ha Binh N,Castillo Mary Joan,Lubkowicz David J,Rowe Sarah E,Millet Yves A,Anderson Cami L,Li Ning,Fisher Adam B,West Kip A,Reeder Philippa J,Momin Munira M,Bergeron Christopher G,Guilmain Sarah E,Miller Paul F,Kurtz Caroline B,Falb Dean. Development of a synthetic live bacterial therapeutic for the human metabolic disease phenylketonuria.[J]. Nature biotechnology,2018,36(9).