Team:TJU China/Description


Roger Tsien is a scientist from our nation who engineered Green fluorescent protein(GFP) in1995, which provides a great contribution to the research of molecular biology.In 2008, Nobel Prize in Chemistry was awarded to Roger Tsien, for “the discovery and development of the green fluorescent protein, GFP".GFP, as the guiding light, has become one of the most important tools in contemporary biological science field. However, He’s not just studying green fluorescent proteins, In the past 10 years, he also engineered a the evolved Far-red fluorescent protein(FP), named small Ultra-Red FP (smURFP) persistently, which can be said not a bit worse than GFP.
The new class of fluorescent protein was developed from an allophycocyanin α-subunit (APCα). Native APC requires a lyase to incorporate phycocyanobilin. The evolved FP, engineered by Roger Tsien, named small Ultra-Red FP (smURFP), covalently attaches biliverdin (BV) without a lyase, and has appropriate excitation/emission peaks, a large extinction coefficient and quantum yield, and comparable photostability to eGFP. What we have done is to make full use of this fluorescent protein as a essential element of our system.
smURFP has 642/670 nm excitation/emission peaks,which means it has advantageous fluorescent intensity, minimize light scattering and absorbance by endogenous biomolecules, which reduces autofluorescence. Also, it uses endogenous chromophores to eliminate O2 necessity and H2O2 production. As what we expected, It can be applied in anaerobic environment, which means that it can be applied to the intestine.What’s more, nontoxic. We can analyze protein structure and find suitable site to perform mutation in order to continue the research of Roger Tsien that has not yet completed, to continue a study which has lasted for ten years by a great scientist of our nation.
As shown above, smURFP is like a torch lighting up some shadow where GFP and other fluorescent peotein cannot reach. It is just like the Promethean fire, which has brought to the world by Mr. Tsien, show us a bright future. That's why we call it "Promethean Fire". Inspired by it, this year we aim to utilizing this novel protein for some new impressive applications.
smURFP can connected with biliverdin and fluoresce which has really good transmission.Therefor we can use this protein to detect biliverdin content.Actually it’s really important for birds,reptiles and egg coloration studies.Birds and reptiles have little biliverdin reductase activity and produce very low amounts of bilirubin.Increased concentrations of biliverdin in serum,bile,or excreta can point to anemia,liver problems and biliary obstruction.biliverdin is also an important pigment of eddshells in several species.Egg coloration studied also require a biliverdin assay to determine biliverdin content in eggshells.
To achieve our aim,we detected the quantitative relationship between protein and pigmen.In the near future,we can easily catch the sign of hepatic disease and occurs of birds and reptiles by using this technology. Because of the high cost of biliverdin,we have also tried to produce biliverdin in our lab.

Part 3.1

What component has the largest amount in human body? Cell or Gene? Neither of the options are right. In fact, it’s microbe! The number of microbes is 10 times larger than that of human cells. Only in our gut, there are more than 100 trillion bacteria, which help the body digest food, produce vitamins to prevent diseases caused by bacteria in the food and stimulate the immune system.
With the in-depth researches, scientists have founded that intestinal microbiota also plays a key role in many chronic diseases and symptoms, such as inflammation and obesity. Some other researchers also suggests one possibility that regulating intestinal microflora might treat diabetes. In addition, newest research has revealed that microbes in the gut can "command” our brains to work. There is an increasing number of evidences indicating that gut flora can really influence our thinking, mood, behavior, and feelings.
At present, the fluorescence system is unable to monitor intestinal bacteria in vivo because of the limitation of wavelength of the fluorescent protein. Our team aimed to create a Real-time fluorescence detection system with the sumURFP.
We searched for some of the most common and important intestinal microorganisms to study their functions, as well as their effects on their host.

Bifidobacterium longum

What component has the largest amount in human body? Cell or Gene? Neither of the options are right. In fact, it’s microbe! The number of microbes is 10 times larger than that of human cells. Only in our gut, there are more than 100 trillion bacteria, which help the body digest food, produce vitamins to prevent diseases caused by bacteria in the food and stimulate the immune system.
More effects about Bifidobacterium are in research by scientists. For example, some researchs show that Bifidobacterium (Bifidobacterium 35624) can inhibit the toxicity of Clostridium difficile on intestinal epithelial HT29 monolayer (in vitro), against Clostridium difficile. In mouse experiments, long Bifidobacterium NCC3001 was found to be involved in vagal anxiolytic effects.Relieve colitis. What’s more, oral bifidobacteria have an obvious inhibitory effect on colitis.

Lactococcus lactis

Lactococcus lactis is a kind of Lactococcus in the most typical, is an important mode of lactic acid bacteria. It is widely used in dairy products and plant products. It is widely used in the food industry and has no pathogenicity to humans and animals. It is considered as a safe food grade microorganism.
In recent years, research on L.lactis molecular biology and mechanism has made significant development. The system has gradually established a series of different uses of the L.lactis gene expression, and successfully expressed many exogenous proteins. Complete genome has been sequenced and therefore, the recombinant Lactococcus lactis has become the focus of food industry, bio-pharmaceuticals and vaccines and is widely used in the field.Applications include recombinant L.lactis for mucosal immunization, recombinant L.lactis expression, transfer of functional proteins, and cytokines.

Some functions and impacts researched:

  1. Recombinant invasive Lactococcus lactis can transfer DNA vaccines either directly to dendritic cells or across an epithelial cell monolayer.
  2. Dextran sulphate sodium colitis in C57BL/6J mice is alleviated by Lactococcus lactis and worsened by the neutralization of Tumor necrosis Factor α.
  3. Lactococcus lactis carrying the pValac eukaryotic expression vector coding for IL-4 reduces chemically-induced intestinal inflammation by increasing the levels of IL-10-producing regulatory cells.

Enterococcus faecium & Enterococcus faecalis

Among several enterococcal species, Enterococcus faecium and Enterococcus faecalis are the two predominant species in the human intestine. The beneficial effects of probiotic Enterococcus spp. in different hosts, such as mice, piglets and humans, to treat various gastrointestinal disorders have been well studied. But they also are the leading cause of highly antibiotic-resistant infections. These bacteria emerged in the last decade of the previous century as one of the primary causes of hospital-acquired infections. The microorganisms isolated from different infections such as endocarditis, bacteremia, and urinary tract infections. Enterococcus faecalis and enterococcus faecium are more prevalent species in such infections. Enterococci have an intrinsically reduced susceptibility to several beta-lactam antibiotics and aminoglycosides; moreover, they may acquire resistance to several antimicrobial agents such as aminoglycosides and beta-lactams via transposes and plasmids. Furthermore, the presence of virulence factors associated with enterococci enhances their pathogenicity. These virulence factors trigger the pathogenicity of the infecting strains by allowing the colonization of the host tissue, invasion of the host tissue, translocation through epithelial cells, and evasion from the host’s immune response. in addition, virulent strains produce pathological changes, either directly by the toxin production or indirectly by inflammation. Therefore, it’s necessary for us to observe these two kinds of intestinal flora for their good and harmful capabilities.

Bacteroides fragilis

Among the anaerobic bacteria that colonize in human and animal intestine tract, Bacteroides fragilis is the predominant bacteria, about 1/4 of the total number of intestinal flora, which is indispensable for maintaining the health of the host. Bacteroides fragilis is a normal mammal intestinal colonized bacteria. It can enhance immunity, prevent intestinal and respiratory diseases, and promote children's body growth and development. It can also regulate the metabolism of some specific substances to change the level of intestinal permeability and restore the normal intestinal flora structure. However, when some part of the body are damaged or have advanced pathological changes, Bacteroides fragilis transposal to be opportunist. In the human colon, Bacteroides fragilis only take up about 1% of the normal flora, whereas cause 60%~90% infection of all the anaerobic bacteria.

Clostridium difficile

Clostridium difficile is the leading cause of infectious diarrhoea in hospitalized patients.One investigation published in The New England Journal of Medicine in 2014 showed that C. difficile was the most commonly reported pathogen (causing 12.1% of all health care–associated infections) in America.
Clostridium difficile is a Gram-positive, spore forming, anaerobic, intestinal bacterium and is the most common cause of antibiotic-associated colitis.Clostridium difficile is the commonest cause of nosocomial diarrhoea in the UK, with C. difficile-associated disease (CDAD) accounting for up to 15% of all diarrhoeal disease associated with antibiotic treatment.

Escherichia coli O157:H7

Shiga-like toxigenic E. coli O157:H7 is a subset of verocytotoxin-producing E. coli (VTEC) and EPEC, as they produce verotoxins like VTEC, also known as shiga toxin-producing E. coli (STEC). It was first recognized as a pathogen because of an outbreak in 1982 of hemorrhagic colitis in the United States. Until now, Shiga-like toxigenic E. coli O157:H7 has caused a large number of foodborne outbreaks worldwide. According to a 2013 report by the United States Department of Agriculture (USDA), E. coli O157:H7 alone cost around 272 million dollars in hospitalizations and corporate food-related losses. EHEC infections remain a significant clinical challenge, antibiotic use is contraindicated due to the ability to exacerbate Shiga toxin (Stx) production and antibiotics and anti-inflammatory drugs increase the risk of developing hemolytic-uremic syndrome.

Citrobacter rodentium

The Gram-negative enteric bacterium C. rodentium is a natural mouse pathogen that produce similar attaching and effacing (A/E) lesions in the intestinal epithelium with human pathogens enteropathogenic (EPEC) and enterohemorrhagic (EHEC). During infection, these bacteria employ surface structures called fimbriae to adhere and colonize the host intestinal epithelium. So C. rodentium infection of mice has been extensively used as an excellent model to study the E. coli infections in vivo. Additionally, the ability of C.rodentium to regulate epithelial barrier integrity, mucosal healing, inflammation, and composition of commensal microbiota makes it a robust model to study the pathogenesis of human intestinal disorders including inflammatory bowel disease, dysbiosis, and tumorigenesis.
The next step has two paths, one is coexpression by express both of the smURFP and HO-1 genes in bacteria.HO-1 can be converted into BV in aerobic bacteria and combined with smURFP to fluoresce in vivo.the other is surface display which are suitable for anaerobe. Surface display technology is a method of obtaining a whole cell catalyst by fusion of the gene encoding the target protein or polypeptide with the gene of the anchor protein, and post-translating and folding the fusion protein to display the target protein on the surface of the host cell wall. By using this technology, we enable smURFP to combine with BV which is added later and fluoresce on the cell surface.
Figure 1. Principle of co-expression construction.
Figure 2. Principle of surface display construction.
When we get this protein, we pay attention to the core function - the nature of the fluorescence. The main advantages have been described in the introduction part, but we still find from the literature that the fluorescence intensity is still not the strongest. The value of EC x QY is approximately equal to eGFP. Moreover, the fluorescence intensity needs to be further improved according to the simulation of our modeling group. That is to say, this protein still has room for improvement. To make the protein better, there are two choices. One is random mutation, which was done by Mr. Tsien, who studied it for 10 years and finally got the target protein. The other is accurate redesign, and precise mutation for active site will greatly shorten the period. It is obvious to us that the second methods is better, which means it require the structure of the protein. Following this idea, we began to analyze the structure, and finally, with the help of PHD in the lab, we did it.Using molecular docking, we simulate combination situation of biliverdin and protein. With the complexes, anyone can conveniently modify the protein based on our work.


[1] Rodriguez EA, Tran GN, Gross LA, Crisp JL, Shu X, Lin JY, Tsien RY. A far-red fluorescent protein evolved from a cyanobacterial phycobiliprotein. Nat Methods. 2016 Sep;13(9):763-9.
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