We built four new parts: [Part: BBa_K3731001], [Part: BBa_K3731002], [Part: BBa_K3731003], [Part: BBa_K3731007] and proved they worked as we expected.
Engineering success is always a very important part in both iGEM and synthetic biology.
Design–Build–Test–Learn (DBTL) cycle is a traditional engineering guidance for synthetic biology.
Following this principle, our iGEM project carried out a series of experiments and researches to achieve engineering success.
Design
One of our team members, who was a member of Prof. Guo Wenjie’s laboratory,
mentioned that the experiments conducted in his lab were promising to treat IBD.
Pro. Guo came up with the idea that inflammatory bowel disease (IBD) is promising to be cured
by long-chain polyP due to its inflammatory-related biological function.
However, long-chain polyP is difficult to produce by chemical synthetic methods.
As a result, we planned to biosynthesize polyP to find a solution of this problem.
Through structure simulation, we determined to utilize PPK1 to produce polyP in E.coli.
Build & Test
In bacteria, polyP is synthesized by polyphosphate kinase (PPK).
PPK1 can lengthen the polymer by using the γ-Pi phosphate bond of ATP,
and its reversible reaction is to synthesize ATP from ADP and Pi.
The ppk1 codes PPK1, which can promote the synthesis (major function) and decomposition (minor function) of polyP
with the residue of ATP. Therefore, we insert ppk1 gene into plasmid PBBR1MCS-2 in order to produce polyP.
Then we transduce the modified plasmid into E.coli BL21 and use synthetic wastewater to culture bacteria.
After 12 hours’ polymerization of phosphorus, bacteria are collected and freeze-dried for preservation.
Therefore, we conducted the above experiment for the first time.
Owing to the lack of experience, our experiment time span is too short
to reflect the overall situation of polyP production efficiency.
Here are our results:
Picture1 values of OD600 against time in the polyphosphorous culture medium in the first experiment.
Picture2 values of OD600 against time in the LB culture medium in the first experiment.
Picture3 values of OD700 against time in the polyphosphorous culture medium in the first experiment.
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.
Learn
At the mid-term meeting in Nanjing, the team of NJMU suggested that we shoud extend the measurement time in the experiment.
What’s more, we asked Prof. Zhi Ding for help.
He proposed that yield is significant in the application of polyP and our yield is not enough for large-scale application.
After literature review and our brainstorm, we figured out that adding vgb and mazE genes into plasmid
is beneficial for bacteria to grow longer and survive tougher environment, thus producing more polyP.
Hence, we adjusted our methods by inserting vgb and mazE genes.
Redesign & Rebuild
In order to achieve a better result, another experiment to produce polyP was conducted.
We improved the experimental method and measured values of OD600 and OD700 for a long time.
Here are our final results:
Picture4 values of OD600 against time in the polyphosphorous culture medium in the third experiment.
Picture5 values of OD600 against time in the LB culture medium in the third experiment.
Picture6 values of OD700 against time in the polyphosphorous culture medium in the third experiment.
Picture7 values of OD against time in the polyphosphorous culture medium in the third experiment.
From the results above, it is not difficult to find that polyP yield increased greatly,
thanks to the insertion of vgb and mazE genes. Therefore, we realized the production of polyP in large scale
and prepared raw materials for our future experiments.
After we obtained the data, we built several models according to the existing literature and modeling methods.
To date, we finished the entire Design–Build–Test–Learn cycle, which is helpful for our final success throughout the whole program.
More information about our experiment
We investigated the advantages of EPVM against normal E.Coli BL21 that only has PBBR1MCS-2,
especially the efficiency of synthesis. In other words, we want to use less bacteria to synthesize more polyP
and acquire more accurate molecular weight of polyP.
So, we drew 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.
We did this experiment for two 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 experiment, we only imported ppk1 with PBBR1MCS-2 named EP,
instead of the whole EPVM(including ppkI, mazE and vgb).
We also made some mistakes in details of measurement.
So, when the first experiment 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 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, which meant a great success for our experiment.
What’s more, We took ppk1 in Citrobacter freundii ATCC 8090 as a comparison and validated its activity through the experiment.
We found that in Citrobacter freundii ATCC 8090, ppk1 can also produce polyP in the E.coli,
which proved our hypothesis. After incubation in the polyphosphorous culture medium for 20 min,
we found that polyP was produced by PPK1, which was proved by ashing and spectrum.
We also measured the curve of OD value CPVM in PA medium. Here is our result:
Picture8 OD measurement of CPVM in the polyphosphorous culture
We compared results of CPVM with results of EPVM, and proved the activity of ppk1 in Citrobacter freundii ATCC 8090.
References
[1] Kyunghye Ahn and Arthur Kornberg, (1990), Polyphosphate Kinase from Escherichia coli, The Journal of Biological Chemistry.