Team:SEHS-China/Engineering
Hay fever, also called allergic rhinitis, causes cold-like signs and symptoms, such as a runny nose, itchy eyes, congestion, sneezing and sinus pressure. But unlike a cold, hay fever isn't caused by a virus. Hay fever is caused by an allergic response to outdoor or indoor allergens, such as pollen, dust mites, or tiny flecks of skin and saliva shed by cats, dogs, and other animals with fur or feathers (pet dander).
The disease, while very common, is not inevitable.If only we could figure out how to do it.The problem, though, is that often by the time we realize we're allergic, the symptoms may be more severe.Therefore, how to quickly determine the situation at an early stage of allergy and leave the area where the allergen may be present becomes very important. To do this, SEHS-China has designed a cell-free system that contains a variety of genetic elements and chemicals, and aims to help patients make the right judgment when an allergy occurs by identifying histamine and releasing special smells and anti-allergic drugs. At the same time, because our products target histamines, our designed sequences can also be used to produce test strips for the freshness of seafood. We're just starting out, and we're facing a lot of problems, but we want to be able to lay the groundwork for using synthetic biology to fight allergies. We sincerely hope that future teams will join us in our research.
Figure 1: These are the results of gel electrophoresis and western blot of CCD4. (a) Those are labeled with CCD4 are results that show the protein of CCD4 are purified, and those are labeled rDAO shows that rDAO are purified. (b) Those are labeled with pET28a-CCD4 are results of CCD4 which have been assembled with pET28a, and those are labeled with rDAO show basepairs of rDAO. (c) This shows the results of colony PCR of hDAO. (d) These are results of PCR of hDAO and pET28a.
Before we can make this cell-free system, we need to make all kinds of proteins, including CCD4, hDAO, LacZ. that need to be wrapped in it.CCD4, the substance that oxidize Carotene into ionol, which produce a special smelling of violet to warn the patient that the histamine level in his or her body in increasing. So it was the first protein that we produced. We inserted the sequence of CCD4 into pET28-a Vector and transformed the plasmid into E. coli BL21. After culturing the edited bacteria, we induced their expression. The expression of CCD4 is good, and the yield is relatively high.
DAO, a chemical that degrades histamine, can significantly reduce histamine levels in the mucous membranes of patients in the early stages of allergy, relieving symptoms and reducing pain. Therefore, THE DAO is a key element in our system. Since our ointment target is human, we first chose hDAO as our production target. However, after inserting the sequence of hDAO into pET28a Vector and converting it into BL21, the induced expression effect was very unsatisfactory. We can hardly see the expression of the product.
Therefore, we speculated that the hDAO sequence might not be suitable for expression in prokaryotes. We might need to do some codon optimization. Codon optimization for hDAO is also an important future work for us.
In order to solve the problem that hDAO could not be effectively expressed, and to verify the central idea of our cell-free system, we adopted rDAO, which had histamine-degrading effect in rats, as a substitute for hDAO.After rDAO was used to repeat the experimental steps of hDAO and induced expression, we found that rDAO could be effectively expressed by BL21.
Therefore, in the subsequent experiments, we will use rDAO as the substance to explain histamine.
LacZ, could be recognized as a reporter gene, is mainly responsible for turn on the riboswitch after sensing the increase of histamine level. The expression of LacZ is also attributed to the expression of BL21. LacZ’s expression as follows:
After designing all the circuits, we purified the proteins expressed by them. Among them, the size of CCD4, rDAO and LacZ is exactly the same as that mentioned in the paper.However, the purification of hDAO is still in a very unsatisfactory state.
In order to verify the effect of rDAO degradation of histamine obtained by us, we carried out the rDAO function examine experiments, and the result supported our expectation. Thus, rDAO can indeed degrade histamine, but the effect is still not obvious. We infer that this is due to the fact that the rDAO we have purified is not very pure. Therefore, the search for a better purification method is one of our very important future works.
Moreover, we express the sensor gene in the j23106 and we put 1 microliter of bacteria solution into each hole of the micro plate. We also add 10 microliter histamine solution into each hole of the micro plate. The concentration of histamine solutions are 0 micro mol/L, 20 micro mol/L, 50 micro mol/L, 100 micro mol/L, 200 micro mol/L, 500 micro mol/L and 1000 micro mol/L. Also, we put 100 microliter LB solution into each hole of the micro plate. Then we put the micro plate into ELIASA and measure the OD600 of the bacteria and the fluorescence of GFP 15 minutes once. So we can use the ratio of these two values to represent the strength of the fluorescence. Then we can draw a graph which show the relationship between histamine concentration and the strength of the fluorescence. After drawing the graph, we can see as the increasing of the histamine concentration, the strength of the fluorescence also increases. Thus, we can say that it has the function of being the histamine sensor. However, during the period that the histamine concentration is low, the graph doesn’t show a linear relation between the histamine concentration and the fluorescence strength. We guess it is because it is hard for histamine to pass the cytomembrane. Since we use the bacteria to show the fluorescence, it will be hard for it to sense the histamine. Thus, we think this proves the significance of the artificial cell. If we replace the bacteria by artificial cell, histamine can easily get into the artificial cell and then be sensed.
With all the parts complete, we proceeded to pack the goods. We adopted the PURE 2.0 system. It's a liposome that isolates our parts from the outside world, but it's also very easily broken down by lipases, which release drugs quickly in the event of an allergy. We got these graphs based on our data.
In conclusion, We successfully produced CCD4, rDAO, LacZ and tested and improved them. And then we wrap them in liposomes, and we make a very sophisticated “early-warning, mitigation, treatment” system. Meanwhile, the application of our plasmids and enzymes in the detection of seafood freshness remains to be studied. At the same time, a better purification method and expression method of hDAO are also urgently needed to be developed.