Team:BUCT-China/Engineering

Due to the new coronavirus in 2020, we can not enter the laboratory in time to complete the experiment. We completed the experimental design of this project in the early stage, and completed part of the experiments in the limited time. We hope to verify the feasibility of our experimental design through part of the experimental content, and do a good foundation for our follow-up work.This page contains the results of our experiments.
We have described our project design ideas (including project background, design ideas, surface display system, etc.) in the design page of Wiki：https://2020.igem.org/Team:BUCT-China/Design

| Preliminary study |

Our team has been concerned about the environmental problems caused by plastic waste for a long time. Based on the study of a strain microbulbifer hydroxyticusire-31 [1], we found that it has the potential to degrade PE.In our previous study, we verified a strain of bacteria (Microbulbifer hydrolyticus) with the potential to degrade PE, and we observed it by electron microscopy
A. there is no Microbulbifer hydrolyticus (a factor simulating the natural environment under UV conditions)
B.degradation of PE by Microbulbifer hydrolyticus(adding Cu + to improve degradation efficiency)

(Fig.1 SEM image of the degradation of PE .（A）The image of the degradation of PE by ultraviolet light after 12 hours .(B)The image of the degradation of PE by Microbulbifer hydrolyticus after 30 days.There are obvious cracks on the PE surface.)

Observing the changes of the surface of PE, it was found that the strain had a certain degradation effect on PE.We conducted a genome-wide analysis of the strain,We believe that yifH is a key enzyme in the whole degradation process.In our previous work, the gene was expressed in E. coli, and the reaction product was detected by GC-MS.It was found that 14-16c alkanes were produced.

（Fig.2 Genome Map）

（Fig.3 GC-MS Analysis method:Samples were detected by GC-MS (Shimadzu GCMS-QP2010, Plus, Ultra, QP2010SE, HS-40, Japan), with a DB-5 column (30m-0.25mm-0.25μm). The temperature program was started at 50 °C for 2 min, then ramped to 300 °C at a rate of 20 °C/min, holding for 7 min at this temperature.）

| Design and construction of degradation part |

In our previous work, we found a key enzyme of PE degradation in this strain (microbulbifer hydroxyticus). We found the target gene yifh by genome sequencing. At the same time, through reading a large number of literature, we believe that surface display technology can be applied to our project, and Bacillus subtilis surface display technology [2] is the most feasible and operational. We designed three main structures: anchor protein, linker peptide and target gene. At the same time, we combined a fluorescent protein GFP to verify the work.
In the work of searching for suitable anchoring proteins, we found that cotg enzymes are widely used in catalysis.

（Fig.4 Anchor protein of CotG[3]）

At the same time, we also found that the linker plays a very important role in the whole system. By comparison, we found that the performance of flexible linker is better than a stiff linker. However, in order to verify, we designed two different linkers simultaneously. Finally, we designed the promoter of cotG and corresponding experimental methods according to some previous experimental ideas ([4] [5] [6]).
Episomal vectors have more replicas, which provides a higher expression level, whilst genome expression is more stable, having no risk of losing target genes. Based on our knowledge, pJS700 (used for episomal expression) and pDG1730[7] (used for genome expression) are the most widely used and reasonable vectors regarding to display of enzyme. Considering that pJS700 is harder to purchase, we decided to chose pDG1730. This also means that we are going to use a double crossover system, which integrates our designed gene to B.subtilis’ genome through a double crossover reaction:

(Fig.5 Methods of integrating target genes into the B. subtilis genome: (A) single crossover and (B) double crossover.)[3]

Our experimental design is shown in the following four figures

| detailed description part |

Construction of recombinant plasmids and bacterial strains

We constructed four plasmid as shown in Figure 6. The plasmid were constructed with DH5α. After constructing the plasmid, we linearized them with ScaI. The linearized plasmids were electroporated into competent B. Subtilis WB800N cells. The sequence between two amyE fragment is supposed to be introduced to amyE locus on genome of WB800N by a double crossover reaction. The integration–positive clones amyE gene is disrupted thus cannot produce amylase. Therefore we identified integration-positive clones by cultivating the bacteria on LB plates containing 1% starch. The integrated clones were expected to yield a blow color after staining with iodine. We further carried out PCR for integration-positive clones to verify the results. We have designed the primers listed below, which will be verified by electrophoresis.

(Fig.6)

Expression and identification of yifH on spore surface

In our experimental design, with the help of GFP, we can easily judge whether the surface display system is successful by observing the fluorescence intensity. At the same time, in order to ensure that the role of the anchoring protein is correct, and that our oxidase is indeed fixed on the outer surface of spores, we have designed a comparative experiment to verify. We set up a control experiment. One group contains anchor protein. If successful, GFP will be expressed on the outer surface of spore along with the target enzyme under the action of anchor protein; the other group does not contain anchor protein, but we also introduce GFP gene. In this way, GFP will not appear on the outer surface of spore, and the possible results can only be inside or not on the spores. Therefore, we only need to compare the fluorescence intensity of the two groups to determine whether our anchoring protein is really effective. (because the fluorescence intensity on the outside of the spore is different from that on the inside (or without fluorescence).We constructed pCYG and pCG to test whether enzyme is anchored on spores’ surface. Cells containing pCYG is expected to express a fused protein cotG-yifH-GFP. This protein will fix at the outer surface due to the anchoring protein cotG and it will emit green florescence with exited with ultra-violate light; Cells harboring pCG is expected to express GFP within cells because there is no anchoring protein fused on GFP. After cultivation of spores, we expect to see fluorescence in both cells.

(Fig.7 Surface display verification experiment)

Measurement of yifH activity

Spores of WB800N harboring pCY1, pCY2, pCYG are cultivated, lyses and purified. We will incubate the spores with PE under 37℃ for different hours. The dry mass of remaining PE will be measured.

| Design and construction of synthesis part |

To further utilized the bio-degraded PE, we decided to establish a novel metabolic pathway in E. coli to convert degraded PE into PHAs. Previous research show that yifH turns PE into straight chain alkane with a length about 14 carbon. Our previous research show that certain P450 enzyme are capable of oxidizing alkane. Hence, we developed the metabolic pathway as follow：

（Fig. 8）

We use CYP153A to hydroxylize alkane and fatty acid, adding oxygen at both end of the molecular. We chose mid-chain alcohol dehydrogenase and aldehyde dehydrogenase that demonstrate desirable activity within ecoli according to other research. We designed the following experimental steps:

To pack up all the 8 enzymes within one cell, we established a dual-plasmid system. The two plasmid are shown below:

（Fig. 9）

Because of the time limit, we can't complete all the experiments. We do some experiments to verify and use them as our part.In the polymerization part, we need to construct long chain alkanes to polyhydroxy fatty acid and synthesize polymers.We believe that the feasibility of the latter pathway is the key to the success of the whole metabolic pathway. Therefore, we tried to construct two plasmids pET-28a (WS / PPF) and pET-28a (WS2 / ACS2) to verify the feasibility of the whole metabolic pathway.At the same time, we used the hydroxyhexadecanoic acid as the substrate to carry out the related verification, and our results were satisfactory.

(Fig.10 metabolic mechanism of fatty acid CoA ligase and acyltransferase)

We expressed pet-28a-WS2 / ACS2 and pet-28a-WS / PPF into E.coli MG1655 At the same time, the related plasmids were also expressed in E.coli MG1655 (fade knockout to exclude the effect of β-oxidation).
1.SDS protein electrophoresis

(Fig.11 SDS protein electrophoresis)

Molecular weight of target protein
WS-DGAT 51.8 kDa
WS2 52.5 kDa
AcsII 61.9kDa
PPF 61.9kDa
Through SDS protein electrophoresis, we can find that by comparing with marker, we can confirm that our gene expression, E.coli has the enzyme we need.

2.Growth curve of E.coli

(Fig.12 Growth curve of E. coli)

After the addition of foreign genes, the ability of substrate assimilation was enhanced, and the growth curve showed satisfactory results; after the fade gene was knocked out, it had a certain impact on the metabolism of bacteria, and the growth and metabolism slowed down, but it still showed that it could grow normally, indicating that the new pathway we constructed was feasible. After knockout, we could exclude the metabolism of fatty acids by the bacteria itself influence.

3.Results of GC-MS with hydroxyhexadecanoic acid as substrate

(Fig. 13 GC-MS diagram of substrate)

We found that the substrate content of E.coli MG1655 (fade knockout, including pet-28a-ws2 / acs2 and pet-28a-ws / PPF) was significantly reduced in E.coli MG1655 (fade knock-out, including pet-28a-WS2 / ACS2 and pet-28a-WS / PPF), which indicated that the fatty acid CoA ligase and acyltransferase metabolic pathways were successfully constructed.

4.GC-MS diagram of products
By GC-MS analysis, we detected a high content of cyclohexadecanolide, which indicated that our fatty acid CoA ligase and acyltransferase metabolic pathways were successfully constructed and reacted with the substrate successfully. Although the polymerization has taken place, it also proves the feasibility of our metabolic pathway to a certain extent. At the same time, it is also a question that we need to explore in the future.

(Fig. 14 GC-MS diagram of products)

| Reference |

[1]Zhongyu Li.Biodegradation of low-density polyethylene by Microbulbifer hydrolyticus IRE-31.Journal of Environmental Management 263 (2020) 110402.
[2]Ping Lin, Haibo Yuan.Progress in research and application development of surface display technology using Bacillus subtilis spores.Applied Microbiology and Biotechnology
[3]Guoyan Zhang.Bacillus subtilis Spore Surface Display Technology: A Review of Its Development and Applications.J. Microbiol. Biotechnol. (2019), 29(2), 179–190.
[4]Wang N, Chang C, Yao Q, Li G, Qin L, et al. (2011) Display of Bombyx mori Alcohol Dehydrogenases on the Bacillus subtilis Spore Surface to Enhance Enzymatic Activity under Adverse Conditions. PLoS ONE 6(6): e21454. doi:10.1371/journal.pone.0021454.
[5]Chaoqun Lian • Yang Zhou • Fan Feng.Surface Display of Human Growth Hormone on Bacillus subtilis Spores for Oral Administration.Curr Microbiol (2014) 68:463–471.
[6]Le H. Duc.Immunization against anthrax using Bacillus subtilis spores expressing the anthrax protective antigen.Vaccine 25 (2007) 346–355.
[7]Youvraj Sohni.Cloning and development of synthetic internal amplification control for Bacillus anthracis real-time polymerase chain reaction assays.Diagnostic Microbiology and Infectious Disease 61 (2008) 471–475.