Overview
We have successfully developed an engineered E. coli-based chronic Lower gastrointestinal bleeding detector. In this biosensor, we have constructed a visual expression system of heme sensitivity, and a simple and portable device for bacterial culture and detection, creating conditions for the practical application of our products. We wanted to prove that these parts could work together to make our project viable as a whole.
Plasmid Construction
Two recombinant plasmids were constructed to visualize the detection of intestinal microbleeds. First, we introduced the heme translocating membrane protein ChuA and the heme binding protein HrtR into the pET28a(+) vector and constructed the pET28a(+)-ChuA-HrtR plasmid. We then ligated the promoter HrtO into pSB1C3-cjBlue (http://parts.igem.org/part:bba_k864404) to construct the recombinant plasmid pSB1C3-HrtO-cjBlue. Among them, the promoter HrtO can be activated with the conformational change of the heme binding monoprotein HrtR.
The plasmid profile of pET28a(+)-ChuA-HrtR
The plasmid profile of pSB1C3-HrtO-cjBlue
Method Development
After transforming two recombinant plasmids, pET28a(+)-ChuA-HrtR and pSB1C3-HrtO-cjBlue, into E. coli BL21(DE3) , we amplified DNA by PCR and sequenced for validation. By exogenous addition of IPTG, the related proteins can be expressed, and the detection and visual expression of heme can be realized.
Experimental Verification
Construction of pET28a(+)-ChuA-HrtR plasmid and pSB1C3-HrtO-cjBlue plasmid
(1) Strain construction
According to the experimental protocol, we first synthesized our desired recombinant plasmid pSB1C3-HrtO-cjBlue by PCR amplification and restriction ligation using pUC19-HrtO and pSB1C3-HrtO-cjBlue (http://parts.igem.org/Part:BBa_K864404 )in iGEM 2019 DNA Distribution Kit Plate 5. Then we transferred two recombinant plasmids pET28a(+)-ChuA-HrtR and pSB1C3-HrtO-cjBlue respectively into E. coli BL21(DE3) .
Transformed colonies of recombinant plasmids pET28a(+)-ChuA-HrtR plasmid and pSB1C3-HrtO-cjBlue
(2) Agarose gel electrophoresis
To verify that our transformed strain contained the correct pET28a(+)-ChuA-HrtR plasmid and pSB1C3-HrtO-cjBlue plasmid, we extracted DNA from the transformed strain and performed PCR and agarose gel electrophoresis. Electrophoresis results indicated that we successfully transfered pET28a(+)-ChuA-HrtR and pSB1C3-HrtO-cjBlue into E. coli BL21(DE3) .
The agarose gel electrophoresis result of plasmid pET28a(+)-ChuA-HrtR
(The HrtO1 band was abnormal and the HrtO2 was normal)
The agarose gel electrophoresis result of plasmid pSB1C3-HrtO-cjBlue
The correct expression of engineering bacteria
(1) Strain construction
Transformed colonies of E. coli containing dual plasmids pET28a(+)-ChuA-HrtR and pSB1C3-HrtO-cjBlue
(2) Agarose gel electrophoresis
The agarose gel electrophoresis results of the strain containing two plasmids
(3)Purification of protein
Figure 1. SDS-PAGE result of supernatant of bacteria broken by ultrasound.
Figure 2. SDS-PAGE results of resuspended precipitate after ultrasonic fragmentation of bacteria.
Hardware
To demonstrate the effectiveness of our biosensors, we have designed a simple bacterial isolation and culture device that can be used in non-laboratory conditions. To ensure that our project can achieve the detection of trace bleeding in the lower gastrointestinal tract. (Please know more about the Hardware)
The hardware flowchart
In our scenario, the user needs to mix the tested faecal sample and load it into a syringe pre-fitted with a specific pore-size filter, followed by extrusion of probiotics into a collection device. In the collection device, we pre-load the medium dry powder, when we want to use, then add water, so that it becomes a liquid medium. Moreover, we also equipped it with a simple oscillating insulation device, which the engineered bacteria can be cultured at a certain temperature oscillating. Finally, we will look for color changes to determine whether heme is present to determine if there is bleeding in the intestine. If the medium appears blue, it proves that the user has a small amount of bleeding in the lower digestive tract.
Hardware entity diagram
So far, we have built and tested the improvised device. It turns out that our engineered bacteria can pass through the membrane and reach the culture below.
Biosafety box
In addition, in order to ensure biosafety, we have also designed a biosafety box with DKU_CHINA to ensure that the engineered bacteria will not leak into the external environment. The hardware was eventually designed by DKU_CHINA and printed using 3D printing technology.
Realization
In order to popularize the public's knowledge about intestinal health, we have jointly developed with other universities a brochure for the Gut Alliance, which aims to guide the public's attention to intestinal health from the perspective of college and high school students, and to help more iGEM intestinal field teams to make the most of our value. (If you want to learn more about our user manual, go to the Gut Alliance page)