iGEM 2019 — Team HUBU-WUHAN

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# Team:HUBU-WUHAN/Notebook

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AWARDS

[Entrepreneurship](https://2019.igem.org/Team%3AHUBU-WUHAN/Entrepreneurship)
[Hardware](https://2019.igem.org/Team%3AHUBU-WUHAN/Hardware)
[Measurement](https://2019.igem.org/Team%3AHUBU-WUHAN/Measurement)
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# Notebook

Document the dates you worked on your project. This should be a detailed account of the work done each day for your project.

### What should this page have?

* Chronological notes of what your team is doing.
* Brief descriptions of daily important events.
* Pictures of your progress.
* Mention who participated in what task.

### Inspiration

You can see what others teams have done to organize their notes:

* [2018 Munich](https://2018.igem.org/Team%3AMunich/Notebook)
* [2014 ATOMS-Turkiye](https://2014.igem.org/Team%3AATOMS-Turkiye/Notebook)
* [2014 Tec Monterrey](https://2014.igem.org/Team%3ATec-Monterrey/ITESM14_project.html#tab_notebook)
* [2014 Kyoto](https://2014.igem.org/Team%3AKyoto/Notebook/Magnetosome_Formation#title)
* [2014 Cornell](https://2014.igem.org/Team%3ACornell/notebook)


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# Team:HUBU-WUHAN/Experiments

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[Demonstrate](https://2019.igem.org/Team%3AHUBU-WUHAN/Demonstrate)
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PARTS

[Parts Overview](https://2019.igem.org/Team%3AHUBU-WUHAN/Parts)
[Basic Parts](https://2019.igem.org/Team%3AHUBU-WUHAN/Basic_Part)
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[SAFETY](https://2019.igem.org/Team%3AHUBU-WUHAN/Safety)

HUMAN PRACTICES

[Human Practices](https://2019.igem.org/Team%3AHUBU-WUHAN/Human_Practices)
[Education & Engagement](https://2019.igem.org/Team%3AHUBU-WUHAN/Public_Engagement)

AWARDS

[Entrepreneurship](https://2019.igem.org/Team%3AHUBU-WUHAN/Entrepreneurship)
[Hardware](https://2019.igem.org/Team%3AHUBU-WUHAN/Hardware)
[Measurement](https://2019.igem.org/Team%3AHUBU-WUHAN/Measurement)
[Model](https://2019.igem.org/Team%3AHUBU-WUHAN/Model)
[Plant](https://2019.igem.org/Team%3AHUBU-WUHAN/Plant)
[Software](https://2019.igem.org/Team%3AHUBU-WUHAN/Software)

[JUDGING FORM ⇗](https://igem.org/2019_Judging_Form?team=HUBU-WUHAN)

# Experiments

Describe the research, experiments, and protocols you used in your iGEM project. These should be detailed enough for another team to repeat your experiments.

Please remember to put all characterization and measurement data for your parts on the corresponding Registry part pages.

### What should this page contain?

* Protocols
* Experiments
* Documentation of the development of your project

### Inspiration

* [2014 Colombia](https://2014.igem.org/Team%3AColombia/Protocols)
* [2014 Imperial](https://2014.igem.org/Team%3AImperial/Protocols)
* [2014 Caltech](https://2014.igem.org/Team%3ACaltech/Project/Experiments)

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# Team:HUBU-WUHAN/Results

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2. Results

# Results

Last year, the iGEM team from Hubei University, accomplished the pathway from waste cartons to isobutanol. Firstly, cartons were smashed and treated with 3% sulfuric acid for 1.5 hours, and then degraded by cellulases into fermentable glucose. Secondly, the glucose was utilized to produce isobutanol by a newly constructed recombinant *Z. mobilis* with a new isobutanol metabolic pathway.

However, there are still some problems. The isobutanol module *Peno-kdcA* led to a low yield of isobutanol, with only 147 mg/L in *Z. mobilis*. And it needed the commercial enzymes for hydrolysis, which caused a lot of waste of energy and high cost.

This year, three modules of the cellulase expression are firstly constructed to make bacteria produce the cellulases, then we can obtain the fermentable glucose from the pretreated paper hydrolyzed with the cell lysate with less commercial enzymes addition. Secondly, a metabolic pathway of the PHB-production is constructed to produce PHB using the fermentable glucose.

## PART 1: Conversion of Waste Paper Cartons into Fermentable Sugars

### Selection of the cellulase genes

To get a more efficient enzyme system and provide more selections, eleven cellulases are selected by searching at NCBI, which are reported to be efficient in different bacteria. Almost all these cellulases are from the Gram-negative bacteria strains[1-4].

![...](https://static.igem.org/mediawiki/2019/4/45/T--HUBU-WUHAN--res1.png)

**Figure 1.** Cellulases selected from different strains.

### Design of cellulase expression module

For a better expression of heterologous cellulase in *Z. mobilis*, selected genes are codon optimized and then synthesized. After then three types of plasmid for the cellulases expression are constructed, which consists of a different strong promoter, the cellulase gene, a FLAG-tag and a strong terminator in the pEZ15A shutter vector. The beta glucanase expression module includes cellulase gene driven by the promoter Peno and terminated by terminator T0117f. The endoglucanase module driven by the strong promoter P*gap* and the strong terminator T0152r. The exoglucanase CBH module driven by the promoter P*eda* and the terminator T1751f.

![...](https://static.igem.org/mediawiki/2019/c/cb/T--HUBU-WUHAN--res2.png)

**Figure 2.** The expression modules of three cellulases.

### Construction of expression modules

Three endoglucanase expression plasmids and five beta glucanase plasmids are constructed using overlap extension PCR cloning, T5 exonuclease assisted cloning and so on. The plasmids that have constructed include the pEZ15A-*Zm*CelA, pEZ15A-*Ct*Cel9D, pEZ15A-*Ec*EG-2, pEZ15A-*Tt*BGL, pEZ15A-*Ct*BGLA, pEZ15A-*Sf*BGL, pEZ15A-*Ru*BGL and pEZ15A-*CHU*>2268. All the recombinant plasmids are transformed individually into *Z. mobilis*, and four recombinant *Z. mobilis* are successfully obtained including the pEZ15A-*Zm*CelA, pEZ15A-*Ct*Cel9D, pEZ15A-*Ec*EG-2, and pEZ15A-*CHU*2268.

![...](https://static.igem.org/mediawiki/2019/d/df/T--HUBU-WUHAN--res3.png)

**Figure 3.** Sequencing results of the recombinant plasmids.

### Western blot and SDS-PAGE detection of cellulases expression

In order to validate the cellulase gene is expressed successfully in *Z. mobilis*, the SDS-PAGE and Western blot are performed. We carry out the SDS-PAGE and Western blot analysis of three cellulases.

### Enzyme activity assay of three cellulases

The activity of three cellulases are quantitatively determined by the DNS method. And three different substrates, the CMC, Salicin and microcrystalline cellulose are used individually to measure the activity of endoglucanase, beta glucanase and [5.6]. By measuring the OD value by Spectrophotometer at 540 nm after the enzymatic reaction, the concentration of the release of reducing sugars from the substrate is obtained from the standard curve of the glucose (Figure 4)[5.6].

![...](https://static.igem.org/mediawiki/2019/4/4f/T--HUBU-WUHAN--res4.png)

**Figure 4.** The standard curve of the glucose.

Compared with the enzyme activity of different recombinant strains, we finally selected the recombinant *Z. mobilis* containing pEZ15A-*Ct*Cel9D as the final scheme of the endoglucanase.

![...](https://static.igem.org/mediawiki/2019/2/2e/T--HUBU-WUHAN--res5.png)

**Figure 5.** The enzyme activity of the constructed Recombinant *Z. mobilis*.

### Pretreatment of the pulp

The primary ingredient of paper cartons is cellulose (ca 63%). Pretreatment with 3% H2SO4 and the enzymatic hydrolysis by cellulases will break down the recalcitrant structure of cartons and release the fermentable mono-sugars.

We plan to compare the concentration of released sugars after the enzymatic hydrolysis by commercial liquid enzymes with and without the whole-cell lysate of the recombinant *Z. mobilis* containing pEZ15A-*Ct*Cel9D addition. However, the experiments are still in progress and the results are not obtained.

![...](https://static.igem.org/mediawiki/2019/3/35/T--HUBU-WUHAN--res6.png)

**Figure 6.** Glucose concentration curve.

![...](https://static.igem.org/mediawiki/2019/4/43/T--HUBU-WUHAN--res7.png)

**Figure 7.** The pulp treating with the liquid enzyme.

## PART 2: Construction of PHB-production Module in *Z. mobilis*

### Establishment of PHB detection method

First, we explored PHB detection with UV and HPLC methods [7]. In the way of UV method, we used chloroform which is volatile and toxic to dissolve PHB. In HPLC method, we tried two approaches to handle dried bacteria. Firstly, we extracted PHB with chloroform and then hydrolyzed with 98% sulfuric acid. Secondly, we directly add 98% sulfuric acid to make an acid digestion of dried bacteria. The results show that these two treatment methods have no significant difference. The standard curves measured by two methods are shown as follow (**Figure 8**, **Figure 9**).

![...](https://static.igem.org/mediawiki/2019/1/1f/T--HUBU-WUHAN--res8.png)

**Figure 8.** PHB standard curve (UV).

![...](https://static.igem.org/mediawiki/2019/4/40/T--HUBU-WUHAN--res9.png)

**Figure 9.** PHB standard curve (HPLC).

From the experimental data, HPLC is more accurate, and from the experimental procedure, HPLC is simpler and safer. Therefore, we chose HPLC to measure PHB.

### Basic Fermentation

In our basic fermentation, we use the promoter Ptet to explore the growth of recombinant strains under the induction of different concentrations of tetracycline[8][9].

![...](https://static.igem.org/mediawiki/2019/6/64/T--HUBU-WUHAN--res10.png)

**Figure 10.** Original design.

![...](https://static.igem.org/mediawiki/2019/d/d4/T--HUBU-WUHAN--res11.png)

**Figure 11.** Growth curve of basic fermentation.

![...](https://static.igem.org/mediawiki/2019/d/d2/T--HUBU-WUHAN--res12.png)

**Figure 12.** PHB production of basic fermentation.

As showed in **Figure 11.**, no significant difference of growth is found in strains containing genes of the expressions of PhbA, *PhbB* and *PhbC*, and wild-type of *Z. mobilis*. However, the production of PHB can be detected in *Z. mobilis* with the help of HPLC. Although the production is very low.

### Metabolic Pathway Optimization

In order to achieve more PHB production, we have designed two methods to optimize its metabolic pathway. The strong promoter P*gap* was selected as the biological element to optimize the metabolic pathway of PHB by increasing the content of precursor acetyl-CoA and co-factor NADPH. We selected several endogenous genes (**Figure 13.**) related to these two factors and enhanced their expression separately (**Figure 14.**).

Glucose-6-phosphate dehydrogenase (ZMO0367) catalyzes the dehydrogenation of glucose 6-phosphate to gluconic acid 6-phosphate with the formation of an NADPH. NAD+ kinase (ZMO1329) catalyzes NADH to NADPH. Dihydrothiocinamide acetyl transferase (ZMO0510) and dihydrothiocinamide dehydrogenase (ZMO0512) are the important components in the pyruvate dehydrogenase system [10][11].

![...](https://static.igem.org/mediawiki/2019/1/14/T--HUBU-WUHAN--res13.png)

**Figure 13.** Four genes in metabolic pathway.

![...](https://static.igem.org/mediawiki/2019/3/31/T--HUBU-WUHAN--res14.png)

**Figure 14.** Five kinds of PHB production plasmids.

![...](https://static.igem.org/mediawiki/2019/b/b9/T--HUBU-WUHAN--res15.png)

**Figure 15.** The growth curve of different recombinant strains.

![...](https://static.igem.org/mediawiki/2019/6/6f/T--HUBU-WUHAN--res16.png)

**Figure 16.** The ethanol production curve of different recombinant strains.

![...](https://static.igem.org/mediawiki/2019/9/98/T--HUBU-WUHAN--res17.png)

**Figure 17.** The PHB production curve of different recombinant strains.

As showed in Fig 15, the recombinant strains of ZM4-P*gap*-1329-CAB, ZM4-P*gap*-0512-CAB and ZM4-P*gap*-0510-CAB grew slowly than the wild-type strain of *Z. mobilis*. However, the recombinant strains of ZM4-P*gap*-CAB and ZM4-P*gap*-0367-CAB grew faster than wild-type *Z. mobilis*. Ethanol production was also determined (Fig 16). The results showed that wild-type strain of *Z. mobilis* produced more ethanol than recombinant strains at the early stage of the fermentation. It can be speculated that the heterogeneous PHB production may inhibit the native ethanol metabolic pathway at the early stage of the fermentation. However, the final PHB production from recombinant strains catch up with or exceed the wild-type strain. Further, the overexpression of ZMO0367 and ZMO1329 were introduced to promote PHB production. The results demonstrated that 6.00% and 7.00% PHB were obtained under the overexpression of ZMO1329 and ZMO0367, respectively, which is higher than the wild-type. We suspect the reason may be that the expression of the ZMO0367 and ZMO1329 increase the amount of NADPH in cells, which is the co-factor of the key enzyme acetoacetyl-CoA reductase (PhbA).

### Metabolic Pathway Optimization

We selected the strain of ZM4-P*gap*-ZMO0367-CAB that with the strongest PHB production ability (**Figure 17.**) to make a fermentation in the carton enzymatic hydrolysis solution.

We used 98% sulfuric acid to dispose samples converting intracellular PHB into crotonic acid. Then HPLC was used to detect the content of crotonic acid in the acid solution.

![...](https://static.igem.org/mediawiki/2019/5/59/T--HUBU-WUHAN--res18.png)

**Figure 18.** The growth curve of different recombinant strains.

![...](https://static.igem.org/mediawiki/2019/e/ef/T--HUBU-WUHAN--res19.png)

**Figure 19.** The ethanol production curve of different recombinant strains.

![...](https://static.igem.org/mediawiki/2019/d/d4/T--HUBU-WUHAN--res20.png)

**Figure 20.** The PHB production curve of different recombinant strains.

As showed in Fig 18 and Fig 19, there is no peak at 28 min in control, while a weak peak was found in the experimental group. This is the peak of crotonic acid. The production of PHB fermented in carton enzymatic hydrolysis solution is 0.378 % (**Figure 20.**).

We successfully produced PHB with the carton enzymatic hydrolysis solution in *Z. mobilis*.

### Reference

1. Zhao et al. **Enzymatic properties of Thermoanaerobacterium thermosaccharolyticum β-glucosidase fused to Clostridium cellulovorans cellulose binding domain and its application in hydrolysis of microcrystalline cellulose**. BMC Biotechnology. 2013, 13:101.

2. Leis et al. **Comparative characterization of all cellulosomal cellulases from Clostridium thermocellum reveals high diversity in endoglucanase product formation essential for complex activity**. Biotechnol Biofuels (2017) 10:240

3. National Bioenergy Center etc. **Heterologous Expression and Extracellular Secretion of Cellulolytic Enzymes by Zymomonas mobilis**. APPLIED AND ENVIRONMENTAL MICROBIOLOGY. Oct. 2010, p.6360–6369

4. P. Thirumalai Vasan etc. **Cellulosic ethanol production by Zymomonas mobilis harboring an endoglucanase gene from Enterobacter cloacae**. Bioresource Technology. 102 (2011) 2585–2589

5. HAROLD C. NEU etc. **The Release of Enzymes from Kwherichia coli by Osmotic Shock and during the Formation of Spheroplasts**. THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 240, No. 9, September 1965

6. T. Okamoto etc. **Cloning of the Acetobacter xylinum cellulase gene and its expression in Escherichia coli and Zymomonas mobilis**. Appl Microbiol Biotechnol (1994) 42:563-568

7. Karr DB, Waters JK, Emerich DW. **Analysis of Poly-beta-Hydroxybutyrate in Rhizobium japonicum Bacteroids by Ion-Exclusion High-Pressure Liquid Chromatography and UV Detection**. Appl Environ Microbiol. 1983;46(6):1339–1344.

8. Juengert, J. R., Bresan, S. and Jendrossek, D. (2018). **Determination of Polyhydroxybutyrate (PHB) Content in Ralstonia eutropha Using Gas Chromatography and Nile Red Staining**. Bio-protocol 8(5): e2748. DOI: 10.21769/BioProtoc.2748.

9. Hiroe A, Tsuge K, Nomura C T, et al. **Rearrangement of Gene Order in the phaCAB Operon Leads to Effective Production of Ultrahigh-Molecular-Weight Poly[(R)-3-Hydroxybutyrate] in Genetically Engineered Escherichia coli>**[J]. 2012, 78(9):3177-3184.

10. Lim SJ, Jung YM, Shin HD, et al. **Amplification of the NADPH-Related Gene zwf and gnd for the oddball biosynthesis of PHB in an E. coli transformant harboring a cloned phbCAB operon**. J Biosci Bioeng, 2002, 93: 543-549.

11. Li ZJ, Cai L, Wu Q, et al. **Overexpression of NAD kinase in recombinant Escherichia coli harboring the phbCAB operon improves poly(3-hydroxybutyrate) production**. Appl Microbiol Biotechnol, 2009, 83:939-947.