Experiment in vitro
Background
What is PolyP
Inorganic polyphosphates (polyP) are linear polymers composed of several to many hundreds phosphate residues. The energy of phosphoanhydride bond in polyP is similar to that in ATP and other nucleoside triphosphates. PolyP have been found in all living cells, from prokaryotes to mammals.
Functions of PolyP
Polyphosphates provide energy storage and a reserve pool of inorganic phosphate, participates in regulation of gene expression, protects cells from the toxicity of heavy metals by forming complexes with them, and participates in channel formation through assembly into complexes with Ca2+and polyhydroxybutyrate and possibly through interaction with channel-forming proteins.
Purpose
1.To synthesize polyphosphate(polyP) in large scale and study the efficiency of polyP production.
2.To investigate the effects of polyP to intestinal epithelial cells under oxidative stress.
Process
Synthesis of PolyP
We insert ppk1 gene into plasmid in order to increase the output of PolyP. Then we transduce the modified plasmid into E.coli BL21 and use synthetic wastewater to culture bacteria. After 12 hours’ polymerization of phosphorus, E.coli are collected and freeze-dried for preservation.
Effects on bacteria and cells
Cell experiments are conducted to construct the intestinal epithelial cell model, so that we can investigate the effects of polyphosphate on intestinal epithelial cells under oxidative stress. To explore the effects of ppk1 on E.coli itself, we measure the growth curves of both modified and unmodified strains in the way of quantitatively describing the content of polyphosphates and the abundance of bacteria.
Apart from the synthesis of polyP, we also investigate the advantages of EPVM against normal E.Coli BL21 that only has PBBR1MCS-2. To this end, we draw the curve of the number of bacteria against culture time (using OD600 as index) and the curve of bacterial function against culture time (polyP production capacity, using OD700 as index). In order to describe the degree of completion of the synthesis of polyP, we use TBE-Urea PAGE assay to estimate the range of molecular weight of PolyP, thus calculating the polymerization degree of polyP.
Citations:
Kulakovskaya, E.V., Zemskova, M.Y. & Kulakovskaya, T.V. Biochemistry Moscow 83, 961–968 (2018).
Andrey Y. Abramov, Cresson Fraley, Catherine T. Diao, Robert Winkfein, Michael A.
Colicos, Michael R. Duchen, Robert J. French, Evgeny Pavlov. PNAS November 13, 2007 104 (46) 18091-18096.
Introduction of IBD
Enteritis is caused by bacteria, viruses, fungi and parasites. There are numerous symptoms of IBD, including abdominal pain, diarrhea, watery stool or mucus pus and blood stool. In a recent report, the morbidity of enteritis is rising in China, posing great threat to people’s health. Several chemical and physical methods have been taken to check for enteritis, among which X-ray barium examination and enteroscopy are two important means. In order to achieve a good therapeutic effect, scientists have utilized different kinds of antibiotics to fight against pathogenic bacteria in the intestine.
Our idea to treat IBD
Since there are many disadvantages of using antibiotic, such as resistance to drugs, it is necessary and urgent to develop an antibiotic-free method to treat IBD. Therefore, we take advantage of biosynthetic polyphosphates(polyP) to improve the microenvironment of intestine. Biosynthetic polyP can enter intestinal epithelial cells by endocytosis, thus inducing cells to express molecular chaperone, which makes it difficult for intestinal epithelial to be damaged by oxidative stress. On the other hand, celluar uptake of polyP can prevent colonization of pathogenic bacteria, thus improving the overall homeostasis of intestinal flora.
Experimental results
1.the curve of the number of bacteria against culture time& the curve of bacterial function against culture time (polyP production capacity)
we investigated the advantages of EPVM against normal E.Coli BL21 that only has PBBR1MCS-2, especially the efficiency of synthesis. In other word, we want to use the less bacteria to synthesize more polyP and the polyP’s molecular weight is more accurate. So, we drew the two kinds of curves and used TBE-Urea PAGE assay to estimate the range of molecular weight of PolyP, thus calculating the polymerization degree of polyP.
(1)the curve of the number of bacteria against culture time
We used spectrophotometer to measure OD600 in different culture time, which is an index to estimate the number of bacteria in culture mediums. Then we drew the curve of the number of bacteria against culture time.
If the OD700 is lower, the more bacteria in the medium.
As a comparison, apart from using EPVM and normal E.Coli BL21, we also used two kinds of culture mediums: LB medium(one kind of common medium, no phosphorus source) and synthetic wastewater medium(High concentration inorganic phosphorus source).
We did this experiment for three times, and every time we finished our experiment, we discussed with Modeling group so that we could improve our experimental protocol and made their model more reasonable and closer to the reality.
The first time Date :July 22nd , 2021
Results are shown as following:
The normal E.Coli BL21 that only has PBBR1MCS-2 grew faster in LB medium at the beginning of the experiment, because its load was smaller. But in synthetic wastewater medium, the result was different. However, the EPVM’s advantage was still shown apparently, it grew faster and more in number, because it could take better advantage of inorganic phosphorus source in the medium and promoted the bacteria to grow faster. The experimental results agreed with the general inference in this respect.
However, Theoretically, both OD700 and OD600 should be constant in the end, fluctuating in a small range around a value, while OD700 is very small, slightly smaller than OD600.
The fitting of the first experiment was good in the early stage, especially in LB medium. But there was no obvious steady state in the late stage in synthetic medium. So we Prolonged culture time(almost two hours) and reduced the interval of measurement time.
The second time Date :July 28th, 2021
Results are shown as following:
The normal E.Coli BL21 that only has PBBR1MCS-2 grew faster in LB medium at the beginning of the experiment, because its load was smaller. the results in two different mediums conformed to general inferences.
But the maximum value of OD600 of the normal E.Coli BL21 that only has PBBR1MCS-2 was kind of small, which was a strange result. We discussed with Modeling group to adjust our plan(test OD600 and OD700 at the same time) and promoted their model.
The third time Date :
Results are shown as following:
(2)the curve of bacterial function against culture time (polyP production capacity)
We used spectrophotometer to measure OD700 in different culture time, which is an index to estimate the residual inorganic phosphorus content in culture mediums. Then we drew the curve of the curve of bacterial function against culture time to measure the polyp production capacity.
If the OD700 is lower, the less residual inorganic phosphorus in medium, the better polyP production capacity it is.
As a comparison, apart from using EPVM and normal E.Coli BL21, we also used two kinds of culture mediums: LB medium(one kind of common medium, no phosphorus source) and synthetic wastewater medium(High concentration inorganic phosphorus source).
We did this experiment for three times, and every time we finished our experiment, we discussed with Model group so that we could improve our experimental protocol and made their model more reasonable and closer to the reality.
The first time Date :July 22nd , 2021
Results are shown as following:
Theoretically, both OD700 and OD600 should be constant in the end, fluctuating in a small range around a value, while OD700 is very small, slightly smaller than OD600.
The fitting of the first experiment was good in the early stage, but there was no obvious steady state in the late stage in synthetic medium. So we Prolonged culture time(almost two hours) and reduced the interval of measurement time.
The second time Date :July 28th, 2021
Results are shown as following:
OD700 fluctuated in a small range around a value very earlier, which was a strange result. We discussed with Modeling group to adjust our plan(test OD600 and OD700 at the same time) and promoted their model.
The third time Date :
Results are shown as following:
2.Estimate the range of molecular weight of PolyP with TBE-Urea PAGE assay (the polymerization degree of polyp)
3.Cell experiments(effects on bacteria and cells)