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 biosynthesize long-chain polyphosphate(polyP) in large scale, which is promising to treat IBD;
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
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.
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.
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 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.
First, with the help of the model of ppk1 created by Modeling group,
we chose ppk1 as our experimental target to adjust the synthesis of polyP.
We did this experiment for three times, and every time we finished our experiment,
we discussed with Modeling group so that we could make their model more reasonable and closer
to the reality and improve our experimental protocol or our parts.
However, in the first two experiments, we only imported ppk1 with PBBR1MCS-2 named EP,
instead of the whole EPVM(including ppk1, mazE and vgb).
We also made some mistakes in details of measurement.
So, when the first two experiments didn't go so well,
we improved our parts and our details of measurement
(such as minimizing the measurement time gradients, extending measurement time and measuring
OD600 and OD700 at the same time as much as possible).
At last, we obtained ideal experimental results and helped Modeling group use the parameters
of the three experiments to compare the effects of this part on the growth of bacteria.
They also created models that were applied for synthetic wastewater medium.
If we get measurement data, we simply plug the data into the model and then we can get various parameters.
(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 OD600 is higher, the more bacteria are in the medium.
As a comparison, apart from using EP, EPVM and normal E.Coli BL21 that only has PBBR1MCS-2,
we also used two kinds of culture mediums: LB medium(one kind of common medium, no phosphorus source)
and synthetic wastewater medium(PA medium, high concentration inorganic phosphorus source).
The first time Date: Apr 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 EP’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(affected by the Competition between bacteria).
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.
At the mid-term meeting in Nanjing, we shared our experimental results with our partner team NJMU-China, they suggested we prolong culture time(almost two hours) and reduce the interval of measurement time.
The second time Date: Jun 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 an unexpected result. We discussed with Modeling group to adjust our plan
(test OD600 and OD700 at the same time) and promoted their model.
We didn’t change our parts at this time, and the OD600 measurement in the first experiment was not bad,
so we used the result of the first experiment as comparison, which was not very precise.
Overall, the bacterial growth model applied well.
The third time Date: Aug 9th, 2021
Results are shown as following:
We used new parts—using EPVM to replace EP. Theoretically, we can get more bacteria and produce much more polyP.
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.
And the maximum value of OD600 of the normal E.Coli BL21 that only has PBBR1MCS-2 was higher but still less than EPVM,
which met our expectations.
Overall, the bacterial growth model applied very well, which was an ideal result.
(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, there is less residual inorganic phosphorus in medium,
and the polyP production capacity is better.
As a comparison, apart from using EP, EPVM and normal E.Coli BL21 that only has PBBR1MCS-2,
we also used two kinds of culture mediums: LB medium(one kind of common medium, no phosphorus source)
and synthetic wastewater medium(PA medium, high concentration inorganic phosphorus source).
The first time Date: Apr 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.
Theoretically, both OD700 and OD600 should be constant in the end, fluctuating in a small range around a value,
while OD700 was very small, slightly smaller than OD600(affected by the Competition between bacteria).
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.
At the mid-term meeting in Nanjing, we shared our experimental results with our partner team NJMU-China,
they suggested we prolong culture time(almost two hours) and reduce the interval of measurement time.
The second time Date: Jun 28th, 2021
Results are shown as following:
We further extended the measure time to make the results more accurate.
OD700 fluctuated in a small range around a value very earlier.
But we didn't know if the yield was up to standard for medicinal use, so we interviewed professor Zhi Ding.
He told us our experimental results were unexpected, which meant the productivity of polyP was not enough for medicinal use.
After pointed out our problems, he further suggested that we could increase the yield of polyP by expanding the size of E.coli.
So we planned to improve our parts. After learning from several literatures, we learned that the ppk1 codes PPK1,
which can promote the synthesis(major) and decomposition(minor) of polyP with the residue of ATP;
the vgb can produce HGB so that we can increase the bacteria’s capacity of carrying oxygen,
which is beneficial to the growth of bacteria; the mazE codes a kind of antitoxin protein,
which can help the bacteria grow longer and produce more polyP. So we will use the whole EPVM(including ppk1, mazE and vgb)
to help the bacteria produce more polyP and grow better.
Apart from improving our parts, we also decided to test OD600 and OD700 at the same time
so that we can observe the connections between OD600 and OD700 more explicitly.
And we can provide more accurate results for Modeling group.
The third time Date: Aug 9th, 2021
Results are shown as following:
Compared with the previous two experiments, the OD700 of EPVM decreased greatly,
especially bigger than EP, which meant the productivity of polyP was ideal and adequate for practical application.
So, the improvement of our parts is successful and meets our expectations.
2. Estimate the range of molecular weight of PolyP with TBE-Urea PAGE assay (the polymerization degree of polyp)
We found that the polyP that are biosynthesized by us is not so long but not so short(about 60 in average).
We found that the major product is P60 and we can improve its productivity by extending culture time
so that we can reduce the pressure and cost of purification.
As a mixture and a polymer, the product needs to be further purified(we can use GPC to separate and purify)
and chemically modified and then it can be applied to the further experiment in vivo.
Conclusion
Compared with the previous two experiments, the OD700 of EPVM decreased greatly, in other word,
the interval between the OD700 in equilibrium and at the beginning is bigger, especially bigger than EP.
The OD600 was also higher, which meant the productivity of polyP was ideal.
The TBE-Urea PAGE statistics show that our biosynthesized polyP has ideal degree of polymerization,
the chain of polyP is long enough(about 60 in average). As a mixture and a polymer,
the product needs to be further purified(we can use GPC to separate and purify) and then we hope to use it to treat IBD.
Attachments
1. LB liquid medium formula
Dissolved 0.5g of yeast powder, 1g of peptone, and 1g of NaCl in water and diluted to 100ml.
2. Synthetic wastewater culture medium formula (100* mother liquor, used sterile water to diluted and mix well)
(1) N source (high pressure steam sterilization)
Dissolved NaCl 0.5g, MgSO4·7H2O 2.36g, and NH4Cl 1.8g in water, and diluted to 100ml.
(2) P source (high pressure steam sterilization)
Dissolved 1.48g of dipotassium hydrogen phosphate trihydrate in water and diluted the volume to 100 ml.
(3) Y source (high pressure steam sterilization)
Dissolved 1g of tryptone and 0.1g of yeast extract powder in water and diluted to 100ml.
(4) Source C (filter sterilization)
Dissolved 3g glucose and 1.5g anhydrous sodium acetate in water and diluted to 100ml.