Team:UCAS
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- Description
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- Sensor
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- Degradation
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- Modelling
Huang Jianyi Cai Ruiling
Every human being hopes to live in a spacious place with enjoyable environment and healthy food to keep fit. We fish have the same dream. Unfortunately, bearing the mission of providing proteins and gourmet for humans, we live in ponds as overcrowded as prison cells, eat nitrogen-rich fishmeal and generate nitrogen-rich waste, and drink nitrogen-rich water. We are expected to grow fast and heavy and be strong and healthy, which is not the case. High nitrogen is toxic to us and may even cost us our lives. We need some help to remove the excess nitrogen in our water.
Lucky as we are, we meet some marvelous friends. They are endowed with the ability to sense the nitrogen concentration in our living water because they intrinsically harbor a nitrogen sensor – NtrC, which is dephosphorylated in high nitrogen environment and phosphorylated in low nitrogen environment. Once phosphorylated under low-nitrogen conditions, NtrC facilitates the binding of sigma-54 factor (σ54) to corresponding σ54-dependent promoters, thus activating these promoters (to know more about the regulatory pathway, click here[Direct to details of sensor pathway]). Our friends, incorporated with a Green Fluorescence Protein (GFP) expressed from a σ54-dependent promoter, can emit green fluorescence when nitrogen concentration is low, meaning “safe”. When we are in dangerous situation where nitrogen concentration is high in water, σ54 no longer binds to promoters and green fluorescence disappears.
Our friends also have the ability to wipe out the excess nitrogen (especially ammonia nitrogen) in our water. Actually, we have two different groups of friends. Our first group of friends want to convert ammonia in our water into nitrogen gas and emit it into air by borrowing a heterogenous pathway. We may call them “gas friends” (to know more about “gas friends”, click here[Direct to description and results page of “gas friends” ]). On the other hand, however, our second group of friends figure to change soluble nitrogen in water into insoluble nitrogen in uric acid and later into sediments using recombinant human xanthine oxidase. We may call them “solid friends” (to know more about “solid friends”, click here).
With the help of our able friends, we live healthily and happily, becoming FIT fish.
To create a biosensor that detects the ammonia in water at low concentrations, we construct a circuit containing a fluoresce reporter, a nitrogen-sensitive promoter together with its regulatory sequences. GlnAp2, GlnHp2 and AstCp are three promoters we tested in our circuits. They are all regulated by NtrC, a general regulatory factor in response to the changes of nitrogen concentrations in extracellular environment. When cells are in a low-nitrogen circumstance, the promoter will be activated and the system will produce green fluoresce signals. (For more details please click here.)
Details
NtrC is a common signal transduction protein in Prokaryote. It functions as a general regulatory factor which is sensitive to the changes of extracellular nitrogen concentrations. GlnAp2, GlnHp2 and AstCp all belong to a type of special promoters. Different from some common promoters like Ptet and Plac, which start transcription through being recognized by RNA polymerase guided by sigma-70 factor, they are recognized by RNP with sigma-54 factor. Besides, only when phosphorylated NtrC binds the regulatory sequences of the promoters locating upstream, can sigma-54 factor be activated and function.
In the normal case, NtrC is expressed by GlnAp1, a sigma-70 factor-dependent promoter, at a low level. When the nitrogen concentration of the environment is below a certain threshold, the nitrogen metabolism of organisms will be constrained, which leads to the increase of acetylphosphate and β-oxoglutarate. With the accumulation of acetylphosphate, the NtrC protein will be phosphorylated by it, and then activate transcription.
GlnAp2 is a promoter that can be activated at low NtrC-p concentrations. It controls the transcription of several important proteins including GlnA, NtrB and NtrC. That is to say, the phosphorylated NtrC can further increase the quantity of NtrC, which amplifies the signal at tremendous speed. Other sigma-54 factor-dependent promoters, for example, GlnHp2 and AstCp, can only be activated in a high level NtrC-p condition.
Of course, there are other proteins assist the signal transduction. Two important ones of them are NtrB and PII.
NtrB, together with NtrC, make up a crucial two-component system in Prokaryote. NtrB has a histine kinase domain, which functions when extracellular nitrogen concentration is rather low. NtrC can bind to it specifically and receive its phosphate group. As mentioned above, the NtrC-p can activate the transcription of NtrB, which indicates another way of the system to amplify the signal. Also notable is that, after phosphorylating the NtrC, NtrB immediately transforms into another conformation functioning as phosphatase. If the whole extracellular environment keeps in low-nitrogen condition, then NtrB in phosphatase conformation will soon turn into kinase conformation again. But if not, the large amount of NtrC-p will return to NtrC state and be inactivated. This is how this system shuts down when cells recover from an extreme circumstance.
Another key protein is PII. Normally, PII binds to NrtB preventing it from react with NtrC. As mentioned before, the pause of nitrogen metabolism causes the accumulation ofβ-oxoglutarate, which can inactive PII and release NtrB.
Generally speaking, the signal transduction of nitrogen deficiency goes as following: lack of nitrogen slow down the intercellular nitrogen circulation, which increases the amount ofβ-oxoglutarate and acetylphosphate. Accumulated β-oxoglutarate release the restrain of PII to NtrB. Increased cetylphosphate first phosphorylates the low level NtrC, and a few NtrC-p activates the transcription of GlnAp2, quickly expressing abundant NtrC and NtrB. These proteins then keep interacting, amplifying the signal with a positive feedback mechanism.
To be short, a low nitrogen threshold provides an input starting the signal transduction, and the system itself quickly gives a significant output.
Description
Our friends also have the ability to wipe out the excess nitrogen (especially ammonia nitrogen) in our water. Actually, we have two different groups of friends. Our first group of friends want to convert ammonia in our water into nitrogen and emit it into air. We may call them "gas friends". On the other hand, however, our second group of friends figure to change soluble nitrogen in water into insoluble nitrogen and later into sediments. We may call them "solid friends"
Let's first talk about our gas friends. They dream of constructing an alien pathway to convert ammonia into hydroxylamine(NH2OH) by ammonia monooxygenase from Anammox, then with ammonia, convert hydroxylamine into hydrazine by the alpha subunit of hydrazine synthase from Anammox, and finally oxidize hydrazine into nitrogen gas by an oxidase. In this case, soluble nitrogen is turned into insoluble nitrogen and our threat is also removed.
Then let's talk about our solid friends. They First of all, when they detect the alarmingly high nitrogen concentration, they begin to absorb more nitrogen from the water and transfer them into purines. Secondly, they can produce a special protein - recombinant human xanthine oxidase, and convert xanthine and hypoxanthine into uric acid precipitation, thus turning soluble nitrogen into insoluble introgen. Combining these two weapons, they alter excessive nitrogen in water, especially nitrogen from ammonia, into nitrogen in sediments with efficiency and proficiency. In this way, we don't have to bear the high concentration of ammonia and can live healthily and happily.
For various reasons, our gas friends died at a young age, but we have to memorize them for their kindness.
Gas group
For this part, our main task is to express the ammonia monooxygenase (AmoA) and the alpha subunit of hydrazine synthase (HZS-α) from Anammox in E.coli, and to select an efficient oxidase that is capable of oxidizing hydrazine into nitrogen gas.
We started with hydrazine oxidation. Since the original hydrazine oxidase(HAO) in Anammox more preferentially oxidizes hydroxylamine rather than hydrazine and the catalytic center is a heme structure, we hoped to use other heme-containing proteins or other oxidases as substitute. We tried 7 proteins, including 3His, degrado, myohemerythrin, UREASE-B, DPS, Pfferritin and CueO, and found that under 37°C and 100mM hydrazine, 3His, degrado, DPS, Pfferritin and CueO can oxidize hydrazine after incubation overnight. Unfortunately, even though Pfferritin was the most effective one among these proteins, it took about an hour to reduce the concentration of hydrazine from 100uM to 50uM, which is far from being efficient in general sense.
Therefore, we decided to put it away for a while, and tackle the other two reactions. First, we tested the ammonia oxidation activity of AmoA-producing E. coli. We added inducer IPTG, and then we incubated the bacteria at 30℃ overnight. Now we hopefully added 1mM NH4Cl in one tube and equivalent of water into another, and hopefully waited for 8 hours. When time was up, we tested the hydroxylamine concentration in both tubes (for hydrazine detection protocol, see notebook), and found that neither of them turned green. Not believing this result, we tried for another time, and another time, and modified culture conditions and tried another time … things did not change and no hydroxylamine was detected.
Now it seemed that the only thing we could do was to express the alpha subunit of HZS. However, one of the teachers in the laboratory told us that he tried but failed, saving us trouble. Well, what about …co-expressing its chaperone? Will that help? Sorry, we had no idea what its chaperone is.
Well, that's how our gas friends perished.
Uric acid producing
In purine catabolism of E. coli, AMP (Adenosine monophosphate) is transformed into hypoxanthine, while GMP is transformed into xanthine. In human, however, hypoxanthine can be oxidized into xanthine and xanthine can be further oxidized into uric acid. These two reactions are catalyzed by the same enzyme: xanthine oxidase. Thus, as long as we produce xanthine oxidase in E. coli, it can also produce uric acid which is slightly soluble in water.
We transformed the recombinant human xanthine oxidase (rhXO) gene into E. coli, expressed it from a lac promoter, and detected uric acid production under different culture conditions using HPLC-MS (with IPTG/without, different culture media). (data being collected)
Nitrogen harnessing
In order to produce more uric acid, we want our E. coli to harness more nitrogen and store it in purines, the precursors of uric acid. Therefore, we cast our sight on purine biosynthesis. In the de novo purine biosynthesis pathway of E. coli, AMP (Adenosine monophosphate) and GMP (Guanosine monophosphate) share a common precursor - IMP (inosine monophosphate), and there are 11 steps to produce IMP from R5P.
We cloned the genes of 5 enzymes in this pathway (figure 2), overexpressed these enzymes and detected the adenine concentration in the culture media using HPLC-MS. All bacteria were cultured in purine-free M9 culture media (for complete protocol, see notebook). The results are shown in figure 3.
It turned out that (purD-coded protein and prs-coded protein) is effective in raising adenine production. To further improve purine production, we introduced point mutation K326Q in purF gene, which is reported to alleviate the feedback inhibition on (enzyme) by AMP and GMP. At the same time, we co-expressed other enzymes with (purD) and (prs) to see whether further improvement in purine production can be achieved.
(data being collected)
Self-sustaining lifestyle
Now that two separate modules are viable, we assemble them together and co-transformed the plasmid bearing rhXO gene and the one bearing genes coding other enzymes. This time, we did not provide extra xanthine and detected uric acid production using HPLC-MS. We contrasted the results with the bacteria not bearing purine production enzymes but enjoying extra xanthine of different concentration.
Complete work
Now we combine our degradation part with our sensor part to construct a complete machine that can be switched on under high nitrogen (ammonia) concentration while stays idle when things get better. We expressed our enzymes under nitrogen-inducible promoters, so that they are only produced when needed.
For Farmers
Publicizing knowledge of ammonia
In order to help more farmers to know the harm of ammonia pollution in ponds, we contacted with the official account of Chinese aquaculture website, and publicized our article though it. The article gained more than 4000 clicks, and fish farmers from all over the china came to share with us their problems or solutions towards ammonia pollution.
Contact with farmers and paying visits to their ponds
Since it is important to and learn about their opinions about ammonia pollution in ponds, we contacted with several farmers in Beijing, paid visits to their ponds and had face-to-face talks with farmers to get more information.
Aquaculture tips brochure
How can we provide more help to fish farmers facing water quality and fish diseases problems? Our team members searched information and wrote a brochure for them. The brochure contained typical problems and tips to improve the water quality in each season, as well as symptoms and solutions for some widespread fish disease in China.
After finishing the brochure, we delivered the brochures to farmers who contacted us online though China. In brief, nearly 400 brochures were sent to twelve cities and counties in China, including Suzhou, Yanan, Yancheng, Jining, Guanzhou and so on.
Bio-safety
Questionnaire about laws on transgene and bio-safety
In order to know more about the opinions on transgene laws from iGEMers and students majoring in synthetic biology, we designed questionnaires and finished the survey. 177 students finished our questionnaire, most of whom were iGEMers or undergraduates and graduates majoring in biology. We found out that laws on bio-safety problems still need to be advertised, since a number of students were not familiar with them and thought it would be helpful to know more. Also, we provided some of the details in the laws, and majority students replied that they were useful to their study and research work.
Video about bio-safety in laborites
This year, UCAS iGEM team collaborated with other nine Chinese iGEM team, making a series of videos which focused on bio-safety to help Chinese students learn more about bio-safety. The topic of our team’s video is personal protective equipment, mainly concerns about the proper use of gloves, lab coat etc in laborites. Here comes our video.
Communication activities
Introduction video to GFP protein and awards
This August, Institute of Physics Chinese Academy of Science, Beijing Association for Science and Technology etc organization held a “3 minutes for science” activity, aiming to collect videos which introduce scientific phenomenon to the public. Our team members made a video to introduce GFP protein and relative knowledge, won the special award and had our video broadcast in the Scientific Carnival in Beijing Olympic Parks.
Beijing-Tianjin meet-up
On Oct 7th and 8th, UCAS iGEM team invited other 4 undergraduate teams and a high school team to our school and held a meet-up, including Zhejiang University, Beijing Normal University, Beijing Institute of Technology, Tianjin University of Science & Technology and The High School Affiliated to Renmin University of China. During the two-day activity, six teams gave presentation of their project, shared ideas and opinions.
Our own We Chat official account
Since WeChat has been widely used in China, we decided to create our own official account to get more people know us and iGEM competition. In the past two months, we have shared introduction of iGEM competition and some details of our team project. To sum up, we received 7731 clicks and more than 3783 persons read our passages.
