Contribution

1 A new intention for improving the performance of starch-based biodegradable plastics

Starch is a soluble white powdery solid and is the major source of energy stored in plants. It is produced by photosynthesizing plants and serves as a storage compound for glucose.

Starch is composed of two substances: amylose, which is a linear polysaccharide, and amylopectin, which is a branched polysaccharide. Here are the differences between amylose and amylopectin (Geeksforgeeks, 2023) (Figure 1, Table 1):

Figure 1 Amylose (Left) and Amylopectin (Right)

Table 1 Difference Between Amylose and Amylopectin

Characteristics	Amylose		Amylopectin
Structure	Linear		Branched
Chain Length	Long		Shorter with branches
Proportion in Starch	20-30%		70-80%
Digestibility	Slower		Faster
Solubility in Water	Lower		Higher
Dissolved in Hot water	Forms a gel		Does not form a gel
Enzyme Activity	More resistant		More easily degraded
Molecular Weight	10^6-10^7 g/mol		10^8-10^9 g/mol

Since amylose and amylopectin have different physicochemical properties, their effects on the performance of starch-based bioplastic products are various. Ma (2009), Andriansyah (2011) and Kumoro (2014) have all compared the performance of bioplastic products produced from raw starches with different ratios of amylose to amylopectin, and found that products with a higher content of amylopectin had better value of tensile strength and elongation at break than products with a lower content of amylopectin. Gabriel (2021) also found the same problem after comparing the performance of different starch-based bioplastic products from previous studies.

According to Sinaga (2014), Utami (2014), Kamsiati(2017), Pradipta (2019), Luqi M. (2021), and Gabriel (2021), it is learned that amylose and amylopectin produce bioplastics with different characteristics. High amylose tends to form crystals that have more substantial mechanical properties than amylopectin, which is amorphous. The crystalline nature of amylose causes starch molecules to become brittle when used as raw material for making bioplastics. The stability of bioplastics was influenced by amylopectin, while amylose affects the compactness of the material. The greater the Amylopectin content, the higher the value of the tensile strength of Biodegradable plastic elongation.

Thus, it is necessary to separate amylose and amylopectin to obtain bioplastics with better results. That means we can also obtain starch-based biodegradable plastics with different performance by adjusting the proportion of amylopectin and amylose in the raw materials.

However, the laboratory mainly obtains high-purity amylose or amylopectin through various isolation and purification methods. Due to the complexity of the process and the high cost of equipment, it is not only difficult to use for large-scale production in factories but also increases the cost of starch-based biodegradable plastics.

By incorporating the traits of synthetic biology, we can modify the starch synthesis pathway in plants using genetic engineering methods to directly produce high-purity amylose and amylopectin. This approach, starting from starch biosynthesis, enables us to adjust the proportion of the two components in raw starch based on requirements for biodegradable plastic products. This practice will allow us to enhance the performance of the end products and to promote the comprehensive application of starch-based biodegradable plastics.

2 Establishment of high-purity amylopectin biosynthesis system

In our project, we have successfully modified the starch synthesis pathway in the plant sweet potato (Ipomoea batatas) using CRISPR/Cas9 gene editing technology to obtain high-purity amylopectin (Figure 2). Firstly, single guide RNA (sgRNA) oligos targeting the IbGBSSI gene was designed and annealed, based on the IbGBSSI gene sequence, and was inserted into the plasmid psgR-Cas9-At to construct the backbone vector of sgRNA (IbGBSSI): psgR-Cas9-sgRNA(IbGBSSI). Secondly, it was combined with the binary vector pCAMBIA1301s to obtain the expression vector of sgRNA (IbGBSSI): psgR-Cas9-sgRNA(IbGBSSI)-p1301s. And then this knockout construct was transformed into embryogenic callus of sweet potato with the method of Agrobacterium-mediated transformation and IbGBSSI-deficient mutants were received after further culturing and screening. After detections of the storage root starch of the IbGBSSI-deficient mutants, it was showed that, compared to the wild-type, the total starch content in the storage roots of the mutants was hardly changed, but the proportion of amylose was significantly reduced to between 0.5% and 0.8% while it was 23.33% of the wild-type (Figure 3). It was indicated that the mutant lines of sweet potato with high-purity amylopectin was obtained by genetic editing.

Figure 2 Schematic representations of CRISPR/Cas9 gene editing construct for the target gene IbGBSSI.

Structural organization of the CRISPR/Cas9 binary vector pCAMBIA1301s used for stable Agrobacterium-mediated transformation in the sweet potato. A. thaliana promoter AtU6 drives expression of the sgRNA. The cauliflower mosaic virus promoter (CaMV 35S) drives expression of the Cas9 gene. Abbreviations: NLS, nuclear localization signal; Nos, Nos terminator.

Figure 3 Amylose content (%) of total starch of root tubers

Obviously, we had successfully established a high-purity amylopectin biosynthesis system. In the future, we will use similar methods to obtain sweet potato lines with high-purity amylose for better use in the production of different performance starch-based biodegradable plastics.

3 Vectors construction

In our project, we have successfully adopted the method of using CRISPR/Cas9 system to target and knock out specific genes in plants, which is rarely used in the iGEM field.

To facilitate the application of this system for the iGEM teams in future, according to Liu et al. (2015), we provide an easy-to-follow procedure for the diverse application of our CRISPR/Cas9 system for targeted gene modifications in plants. We also offer a range of solutions in the process, from the design of sgRNAs and vector construction to the detection of specific gene modifications and the selection of gene-edited plants. We hope that the principles described here to customize the versatile CRISPR/Cas9 system can help more iGEMers for gene editing in plants.

The main steps are as follows:

(1) Selection of sgRNA targets

The appropriate sgRNA targets for the interest gene will be selected using the online sgRNA design tools. The following are some web-based sites for the selection of sgRNA targets:

1. http://www.plantsignal.cn/CRISPR/crispr_primer_designer.html
2. http://www.genome-engineering.org/crispr/?page_id=41
3. https://www.dna20.com/eCommerce/cas9/input
4. http://www.genome.arizona.edu/crispr/index.html

(2) Design of the gRNA oligonucleotides

Once a 20-nt target site is selected, a pair of DNA oligos can be synthesized as follows,

Forward oligo: 5’-gattGNNNNNNNNNNNNNNNNNNNN-3’

Reverse oligo: 5’-aaacNNNNNNNNNNNNNNNNNNNNC-3’

(3) Construction of the backbone vector

1. Phosphorylate and anneal each pair of oligos.
2. Digest 0.5 μg of psgR-Cas9-At with BbsI for at least 2 h at 37 ºC.
3. Ligate the BbsI-digested vector with the oligo duplex.
4. Transform E. coli with the ligation product.
5. Identify positive clones by colony PCR.

(4) Construction of the plant expression backbone vector

1. Digest the pCAMBIA1301S vector and the backbone vector with HindIII and EcoRI in separate reactions at 37 ºC for 2 h.
2. Run the reactions on a 1 % agarose gel, cut the digested band of the backbone vector and the band of linearized pCAMBIA1301S.
3. Set up the ligation reaction incubate at 16 ºC for 2 h.
4. Transform the ligation reaction into DH5 alpha competent cells and plate cells on LB plates containing 50 lg/mL kanamycin for selection in this case.
5. Identify positive clones by colony PCR.
6. Purify plasmid from the culture of the positive clones.
7. Transform the plasmid into Agrobacterium tumefaciens LB4404 using the freeze–thaw (Weigel et Glazebrook, 2006) or any other equivalent method.

(5) Plant transformation

When transformed Agrobacterium tumefaciens is obtained, plant can be transformed using the method of Agrobacterium-mediated transformation, of which the procedure details vary in different plants.

4 Reference:

Ardiansyah R. 2011. Pemanfaatan pati umbi garut untuk pembuatan plastik biodegradable. Universitas Indonesia : Depok.

Gabriel A A. 2021. Tensile Strength and Elongation Testing for Starch-Based Bioplastics using Melt Intercalation Method: A Review

Geeksforgeeks (2023) Difference between amylose and amylopectin. https://www.geeksforgeeks.org/amylose-vs-amylopectin/

Kamsiati E, Herawati et dan Purwani E Y. (2017). Potensi pengembangan platik biodegradable berbasis pati sagu and ubi kayu di Indonesia. Jurnal Litbang Pertanian, 36 (2): 67-76.

Kumoro AC, Purbasari A. 2014. Sifat mekanik dan morfologi plastik biodegradable dari limbah tepung nasi aking dan tepung tapioka menggunakan gliserol sebagai plasticizer. Semarang: Universitas Dipenogoro.

Liu WS, Zhu XH, Lei MG et al. (2015) A detailed procedure for CRISPR/Cas9-mediated gene editing in Arabidopsis thaliana. Sci. Bull., 60(15): 1332–1347

Luqi M et Rahman ED. (2021) The effect of amylopectin on plastic quality. ejurnal. bunghatta. ac. id,18 (4): Summary Executive Jurusan Teknik Kimia Wisudawan ke 76

Ma FC. 2009. Preparation and properties of glycerol plasticized-pea starch/zinc oxide-strach bionanocomposites. Carbohydrate Polymers, 75: 472-478.

Pradipta R A. 2019. Potensi manufacture of biodegradable plastics from cassava peel strach and garut sweet potato strach with melt intercalation methods in supporting of SDG’s. Proceeding Book 7th Asian Academic Society International Conference 2019. ISBN: 978-602-61265-5-9

Sinaga R F, Ginting G M, Ginting M H S et Hasibuan . 2014. Pengaruh penambahan gliserol terhadap sifat kekuatan tarik dan pemanjangan saat putus bioplastik dari pati umbi talas. Jurnal Teknik Kimia USU, 3 (2): 19-24.

Utami M R, Latifah et Widiarti N. 2014. Sintesis plastik biodegradable dari kulit pisang dengan penambahan kitosan dan plasticizer gliserol. Indonesian Journal of Chemical Science, 3 (2): 163-167.

Weigel D et Glazebrook J. (2006) Transformation of Agrobacterium using the freeze-thaw method. CSH Protoc. doi:10.1101/pdb.prot4666