iGEM 2023 — Team shanghai-biox

{% extends "layout.html" %} {% block title %}Notebook{% endblock %} {% block lead %}Document the dates you worked on your project. This should be a detailed account of the work done each day for your
project.{% endblock %} {% block page\_content %}

# Notebook

## **Summary**

---

Establishment of engineered sweet potato is a time-consuming task as the seedling regeneration and the accumulation of starch in the storage roots require a relatively long period of time.
The following is a record of the process of our project experiment. For detailed operation procedures and experimental results, please refer to the
[Protocols](https://2023.igem.wiki/shanghai-biox/protocols%20) and [Engineering](https://2023.igem.wiki/shanghai-biox/engineering) Success pages.

## **Notebook**

#### [1 March](#Header1)

#### [2 April](#Header2)

#### [3 May](#Header3)

#### [4 June](#Header4)

#### [5 July](#Header5)

#### [6 August](#Header6)

## **1 March**

1. Embryogenic calli induction of sweet potato
2. sgRNA target selection
3. sgRNA design
4. sgRNA oligos synthesis

## **2 April**

* Embryogenic calli proliferation of sweet potato

  ![](https://static.igem.wiki/teams/4887/wiki/images/notebook/01.png)

  **Fig. 1 Embryogenic callus culture of sweet potato**
* Construction of the backbone vector

  ![](https://static.igem.wiki/teams/4887/wiki/images/notebook/02.png)

  **Fig. 2 Digestion result of psgR-Cas9-At**

  ![](https://static.igem.wiki/teams/4887/wiki/images/notebook/03.jpg)

  **Fig. 3 E. Coli transformed with backbone vector of sgRNA (IbGBSSI)**

  ![](https://static.igem.wiki/teams/4887/wiki/images/notebook/04.jpg)

  **Fig. 4 PCR product of sgRNA (IbGBSSI)**
* Construction of the plant expression vector

  ![](https://static.igem.wiki/teams/4887/wiki/images/notebook/05.jpg)

  **Fig.5 Digestion result of psgR-Cas9-sgRNA and pCAMBIA1301s**
* Agrobacterium tumefaciens transformation

  ![](https://static.igem.wiki/teams/4887/wiki/images/notebook/06.jpg)

  **Fig. 6 A. tumefaciens transformed with the vector psgR-Cas9-sgRNA(IbGBSSI)-p1301s**

  ![](https://static.igem.wiki/teams/4887/wiki/images/notebook/07.jpg)

  **Fig. 7 PCR result of A. tumefaciens transformed with psgR-Cas9-sgRNA(IbGBSSI)-p1301s**
* Agrobacterium-mediated transformation for sweet potato

  ![](https://static.igem.wiki/teams/4887/wiki/images/notebook/08.png)

  **Fig. 8 Embryogenic callus co-culture with A. tumefaciens transformants (A) and selection of positively transformed callus with hygromycin (B)**
* Screening and selection of calli mutant

## **3 May**

* Regeneration and sub-culture of transgenic sweet potato seedlings
* GUS detection for regenerated seedlings

  ![](https://static.igem.wiki/teams/4887/wiki/images/notebook/09.png)

  **Fig. 9 Results of GUS staining**
* PCR detection for regenerated seedlings

  ![](https://static.igem.wiki/teams/4887/wiki/images/notebook/10.jpg)

  **Fig. 10 Detection results of PCRs for gene Hyg and gene Cas9.**
* Transplantation of transgenic seedlings

## **4 June**

* Literature review

## **5 July**

* Determination of chlorophyll content in leaves

  ![](https://static.igem.wiki/teams/4887/wiki/images/notebook/11.png)

  **Fig. 11 chlorophyll contents of the transgenic lines**

## **6 August**

* Storage roots harvest

  ![](https://static.igem.wiki/teams/4887/wiki/images/notebook/12.png)

  **Fig. 12 Phenotypes of the IbGBSSI-knockout lines planted in greenhouse**
* Determination of the expression level of IbGBSSI in storage roots

  ![](https://static.igem.wiki/teams/4887/wiki/images/notebook/13.png)

  **Fig. 13 Q-PCR result of the relative expression level of IbGBSSI in root tubers**
* Extraction of total starch in storage roots
* Starch analysis of transgenic sweet potato storage roots

  + Qualitative detection of the starch components by iodine staining

    ![](https://static.igem.wiki/teams/4887/wiki/images/notebook/14.png)

    **Fig. 14 Component detection of total starch by iodine staining**
  + Quantitative detection of the starch composition

    ![](https://static.igem.wiki/teams/4887/wiki/images/notebook/15.png)

    **Fig. 15 Amylose content (%) of total starch of root tubers**

{% endblock %}

{% extends "layout.html" %} {% block title %}Engineering Success{% endblock %} {% block lead %}Demonstrate engineering success in a part of your project by going through at least one iteration of the
engineering design cycle.{% endblock %} {% block page\_content %}

# Engineering Success

## **Overview**

---

Starch is a natural polymer composed of amylose and amylopectin, and is an excellent raw material for biodegradable plastics. The properties of starch-based bioplastics are influenced by the
ratio of amylose to amylopectin in the raw starch so that starch-based bioplastics with different properties can be obtained by adjusting this ratio, which requires high-purity amylose and
amylopectin.

To reach this goal, the project aims to modify the starch synthesis pathway in the plant sweet potato (Ipomoea batatas) using CRISPR/Cas9 gene editing technology to obtain high-purity
amylopectin. 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. Thirdly, 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.

It was indicated that sweet potato lines with high-purity amylopectin was obtained by genetic editing; 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.

## **Engineering**

#### [1 Vector construction](#Header1)

[1.1 Design and synthesis of sgRNA](#Header2)
[1.2 Construction of the backbone vector](#Header3)
[1.3 Construction of the expression vector](#Header4)
[1.4 Agrobacterium tumefaciens transformation](#Header5)

#### [2 Genetic transformation of sweet potato](#Header6)

[2.1 Induction and culture of embryogenic callus](#Header7)
[2.2 Agrobacterium-mediated transformation](#Header8)

#### [3 Verification of transgenic sweet potato plants](#Header9)

[3.1 GUS detection](#Header10)
[3.2 PCR detection](#Header11)
[3.3 Determination of chlorophyll content in leaves](#Header12)
[3.4 Determination of the expression level of IbGBSSI in storage roots](#Header13)

#### [4 Starch analysis of transgenic sweet potato root tubers](#Header14)

[4.1 Qualitative detection of the starch components](#Header15)
[4.2 Quantitative detection of the starch composition](#Header16)

#### [5 Future work](#Header17)

#### [6 Reference](#Header18)

## **1 Vector construction**

### **1.1 Design and synthesis of sgRNA**

The sweet potato granule-bound starch synthase I (IbGBSSI) gene (Accession Number: AB071604) contains nine exons and CRISPR/Cas9 targets the first one. The appropriate sgRNA targets for
the gene IbGBSSI were selected using the online sgRNA design tools and the available I. batatas draft genome sequences (Yang et al., 2017). The selected sgRNA is located 70 bp downstream
of the start codon, with a total sequence length of 27 bp (Fig. 1). (The selected sgRNA will be cloned from the target gene.)

sgRNA oligos were obtained by annealing with the designed primers following:

Oligo 1 (GBSSI-sgRNA1): 5’-GTGGGGTTGGGTCAATTAGCCCTGAGGAGC-3'

Oligo 1 (GBSSI-sgRNA2): 5’-AACTGGTGGACTTGGAGATGTTCTTGGAGG-3’

![](https://static.igem.wiki/teams/4887/wiki/images/engineer/01.png)

**Fig. 1 The target region of gene IbGBSSI and the location of the gRNA**

Exons are shown as square frames and surrounding introns appear as lines. sgRNA and PAM are highlighted in yellow and green, respectively.

### **1.2 Construction of the backbone vector**

The plasmid psgR-Cas9-At were digested (Fig. 2) and the products were ligated with sgRNA oligos using T4 ligase to obtained the backbone vector of sgRNA (IbGBSSI): psgR-Cas9-
sgRNA(IbGBSSI). 1/2 of the DNA was then transform into bacteria and incubate in LB for 45 min, and was plated in LB containing Ampicillin (Fig. 3). The obtained clones were validated by
PCR with primers M13F/oligo2 (The sequence of M11F was: 5’-TGTAAAACGA CGGCCAGT-3’) (Fig. 4).

![](https://static.igem.wiki/teams/4887/wiki/images/engineer/02.png)

**Fig. 2 Digestion result of psgR-Cas9-At**

![](https://static.igem.wiki/teams/4887/wiki/images/engineer/03.jpg)

**Fig. 3 E. Coli transformed with backbone vector of sgRNA (IbGBSSI)**

![](https://static.igem.wiki/teams/4887/wiki/images/engineer/04.jpg)

**Fig. 4 PCR product of sgRNA (IbGBSSI)**

### **1.3 Construction of the expression vector**

The validated backbone vector of sgRNA (IbGBSSI) was digested by EcoR I & Hind III (Fig. 5) and inserted into the corresponding sites of the binary vector pCAMBIA1301s, harbouring the
Hygromycin B resistance gene HygR and the reporting gene GUS, to obtain the expression vector of sgRNA (IbGBSSI): psgR-Cas9-sgRNA(IbGBSSI)-p1301s.

![](https://static.igem.wiki/teams/4887/wiki/images/engineer/05.jpg)

**Fig. 5 Digestion result of psgR-Cas9-sgRNA and pCAMBIA1301s**

The expression vector of sgRNA (IbGBSSI), harbouring the single gRNA cassette with the Cas9 endonuclease, were constructed. The U6 promoter from Arabidopsis (pAtU6), was used to drive
the sgRNA expression. The Arabidopsis ubiquitin-1 promoter (AtUBQ1) was used to control Cas9 expression (Fig. 6).

![](https://static.igem.wiki/teams/4887/wiki/images/engineer/06.png)

**Fig. 6 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.

### **1.4 Agrobacterium tumefaciens transformation**

The expression vector of sgRNA (IbGBSSI) was successfully transferred into Agrobacterium tumefaciens LB4404 by freeze-thaw method. The positive transformants containing were selected and
cultured on solid YEB medium plates containing antibiotics of rifampicin, chloramphenicol and kanamycin (Fig. 7). The selected clones were validated by PCR later with primers M13F/oligo2
(Fig. 8).

![](https://static.igem.wiki/teams/4887/wiki/images/engineer/07.jpg)

**Fig. 7 A. tumefaciens transformed with the vector psgR-Cas9-sgRNA(IbGBSSI)-p1301s.**

![](https://static.igem.wiki/teams/4887/wiki/images/engineer/08.jpg)

**Fig. 8 PCR result of A. tumefaciens transformed with psgR-Cas9-sgRNA(IbGBSSI)-p1301s**

## **2 Genetic transformation of sweet potato**

The stable transformation of sweet potato was performed using the protocol of Yang et al (2011) .

### **2.1 Induction and culture of embryogenic callus**

We took the axillary buds from the pre-prepared sterile sweet potato variety B23 seedlings, inoculated the meristematic tissue onto MSD solid medium, and successfully induced embryogenic
callus (Fig. 9). Afterwards, these embryogenic calli were transferred to new MSD solid medium and LCP liquid medium successively for propagation (Fig 10).

![](https://static.igem.wiki/teams/4887/wiki/images/engineer/09.png)

**Fig. 9 The process of sweet potato embryogenic callus induction**

![](https://static.igem.wiki/teams/4887/wiki/images/engineer/10.png)

**Fig. 10 Embryogenic callus culture of sweet potato**

### **2.2 Agrobacterium-mediated transformation**

When enough of sweet potato embryogenic callus ware obtained, they were infected with the A. tumefaciens transformants containing the vector for CRISPR-knockout expression by co-culture
(Fig. 11A). After selection with hygromycin, positive transformed calli were obtained (Fig. 11B). These calli were further cultured to obtain transgenic sweet potato seedlings (Fig. 12).

![](https://static.igem.wiki/teams/4887/wiki/images/engineer/11.png)

**Fig. 11 Embryogenic callus co-culture with A. tumefaciens transformants (A) and selection of postively transformed callus with hygromycin (B)**

![](https://static.igem.wiki/teams/4887/wiki/images/engineer/12.jpg)

**Fig. 12 Seedlings of IbGBSSI-knockout lines after preliminary selections**

## **3 Verification of transgenic sweet potato plants**

### **3.1 GUS detection**

he GUS gene, is a commonly used reporter gene harboured in pCAMBIA1301s. Its expression product β-glucuronidase is a hydrolase that can catalyze the hydrolysis of many β-glucoside
esters. It can decompose X-Gluc into blue substances, to observe the expression of foreign genes in transgenic plants and identify transgenic plants.

After the regenerated seedlings grew leaves, we conducted GUS staining on these transgenic plants and preliminarily screened two successfully transformed sweet potato lines, designated
as 23216004 and 23216005 (Fig. 13).

![](https://static.igem.wiki/teams/4887/wiki/xiugai-2012/eng-13-results-of-gus-staining.png)

**Fig. 13 Results of GUS staining**

### **3.2 PCR detection**

Genome DNA of these two transgenic lines was extracted from leaves of these two regenerated seedlings. PCR detection on the genomes was performed by using two pairs of primers which were
designed based on the Cas9 protein gene and the hygromycin resistance gene (HygR), respectively. As the result, the transgenic lines 23216004 and 23216005 were further validated (Fig.
14).

The sequences of the two pairs of primers were as bellow:

* CAS9-F: 5’-ATGGACTATAAGGACCACGACGG-3’;
* CAS9-R: 5’-TTGTCGCCTCCCAGCTGAGACAG-3’
* HygR-F: 5’-Atgaaaaagcctgaactcac-3’;
* HygR-R: 5’-ctatttctttgccctcggac-3’

Subsequently, these two transgenic lines and the wild-type B23 were planted with the method of cuttage in an experimental greenhouse to harvest starch-rich tubers.

![](https://static.igem.wiki/teams/4887/wiki/images/engineer/14.jpg)

**Fig. 14 Detection results of PCRs for gene Hyg and gene Cas9**

### **3.3 Determination of chlorophyll content in leaves**

During the cultivation process, there is no visible difference in the above-ground phenotype between the transgenic line and the wild type. To confirm whether the gene knockout has an
impact on the physiology of the transgenic line, we measured the chlorophyll content in their leaves.

Each transgenic line and wild type were sampled from three different plants, and the samples were prepared into chlorophyll solutions. The absorbance values of the solutions at
wavelengths of 663 nm and 645 nm were determined using spectrophotometry method, designated as OD663 and OD660, respectively. Afterwards, the following formulas were applied to calculate
the concentrations of chlorophyll a, chlorophyll b, and total chlorophyll for each sample:

Chl a = 12.7 x OD663 - 2.69 x OD646 (1)

Chl b = 22.9 x OD646 - 4.68 x OD663 (2)

Chl TOT = 8.02 x OD663 + 20.2 x OD646 (3)

In the formulas,

Chl a: Content of chlorophyll a (ug/L);

Chl b: Content of chlorophyll b (ug/L);

Chl TOT: Total content of chlorophyll a and chlorophyll b (ug/L);

According to the results, it could be seen that both transgenic lines have chlorophyll A and B contents similar to the wild type, at around 0.6000 mg/g and 0.2500 mg/g respectively
(Table 1; Fig. 15). It showed that the chlorophyll contents of the transgenic lines were basically consistent with that of the wild type, ruling out the impact of gene-knockout on the
sweet potato starch synthesis pathway.

**Table 1 chlorophyll contents of the transgenic lines**

|  |  |  |  |  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| Line No. | mass | OD663 | OD645 | congtent of chl a (mg/g) | | congtent of chl b (mg/g) | | congtent of total chl (mg/g) | |
|  | mean+SD |  | mean+SD |  | mean+SD |
| B23 | spl1 | 0.4722 | 2.6045 | 1.0812 | 0.6389 | 0.6085+0.0304 | 0.2662 | 0.2535+0.0127 | 0.9186 | 0.8749+0.0437 |
| spl2 | 0.4571 | 2.4012 | 0.9968 | 0.6085 | 0.2535 | 0.8749 |
| spl3 | 0.4623 | 2.3071 | 0.9577 | 0.5781 | 0.2408 | 0.8311 |
| 23216004 | spl1 | 0.4812 | 2.6173 | 1.0909 | 0.6298 | 0.5998+0.0300 | 0.2646 | 0.2520+0.0126 | 0.9078 | 0.8646+0.0432 |
| spl2 | 0.4931 | 2.4266 | 1.0115 | 0.5698 | 0.2394 | 0.8213 |
| spl3 | 0.4786 | 2.4792 | 1.0334 | 0.5998 | 0.2520 | 0.8646 |
| 23216005 | spl1 | 0.4802 | 2.6184 | 1.0828 | 0.6318 | 0.6018+0.0301 | 0.2612 | 0.2487+0.0124 | 0.9063 | 0.8631+0.0432 |
| spl2 | 0.4922 | 2.4282 | 1.0041 | 0.5717 | 0.2363 | 0.8200 |
| spl3 | 0.4768 | 2.4760 | 1.0239 | 0.6018 | 0.2487 | 0.8631 |

![](https://static.igem.wiki/teams/4887/wiki/images/engineer/15.png)

**chlorophyll contents of the transgenic lines**

### **3.4 Determination of the expression level of IbGBSSI in storage roots**

Two months after transplantation, the storage roots of the transgenic lines were harvested. It showed that the number and size of the root tubers were largely consistent with the wild
type (Fig. 16).

![](https://static.igem.wiki/teams/4887/wiki/images/engineer/16.png)

**Fig. 16 Phenotypes of the IbGBSSI-knockout lines planted in greenhouse**

RNA was extracted from the fresh root tubers and cDNA was obtained through reverse transcription later. Then, the relative expression level of the gene IbGBSSI was determined with the
method of Quantitative Real-time PCR with the root-tuber cDNA as templates. The result showed that the relative expression level of IbGBSSI in storage roots of the transgenic lines
(0.1063 and 0.2407) was much lower than that of the wild type (1.0000) (Fig. 17). It revealed that the knock-out of IbGBSSI in the pathway of starch synthesis was successful.

![](https://static.igem.wiki/teams/4887/wiki/images/engineer/17.png)

**Fig. 17 Q-PCR result of the relative expression level of IbGBSSI in root tubers**

## **4 Starch analysis of transgenic sweet potato root tubers**

Freshly harvested sweet potato tubers were cleaned, peeled, and sliced into small pieces. Starch was extracted from these pieces for qualitative and quantitative detection afterwards.

### **4.1 Qualitative detection of the starch components**

When exposed to iodine, amylose appears blue, while amylopectin appears reddish brown or purple red. Therefore, the component qualitative detection of the total starch from the
transgenic lines was performed. As the result, the total starch of the transgenic lines appears reddish brown while that appears blue of the wild type (Fig. 18). It indicated that the
total starch of the transgenic lines was composed mainly of amylopectin.

![](https://static.igem.wiki/teams/4887/wiki/xiugai-2012/eng-18-component-detection-of-total-starch-by-iodine-staining.png)

**Fig. 18 Component detection of total starch by iodine staining**

### **4.2 Quantitative detection of the starch composition**

Firstly, standard starch solutions with different gradients of amylose content were prepared (Table 2) and their absorbance values at a wavelength of 620nm, OD620, were obtained (NY/T
2639-2014). Subsequently, using the absorbance values as the vertical axis and the amylose content as the horizontal axis, the standard curve representing the linear relationship was
plotted between the content of branch starch and OD620 and the following calculation formula was fitted (where y represents the content of amylose in total starch (%), and x represents
OD620) (Fig. 19):

y = 197.47x - 14.829, R2 = 0.9977 (4)

**Table 2 OD620 values of standard solutions of starch**

|  |  |
| --- | --- |
| OD620 | Content of amylose (%) |
| 0.092 | 0 |
| 0.126 | 10 |
| 0.170 | 20 |
| 0.219 | 30 |
| 0.272 | 40 |
| 0.377 | 60 |
| 0.480 | 80 |
| 0.589 | 100 |

![](https://static.igem.wiki/teams/4887/wiki/images/engineer/19.jpg)

**Fig. 19 Standard curve for amylose content (%) of total starch**

Later, starch solutions were prepared respectively from total starch extracted earlier from storage roots of the two genotypes and the wild type and their OD620 values were determined,
with three replicates for each line. After plugging the obtained OD620 values into formula (4), the amylose content was quantified in each sample.

Compared to the amylose content of the wild type (23.3306%), the amylose content in the total starch of the two transgenic lines was only 0.5657% and 0.7613%, respectively (Table 3, Fig.
20). According to the study by Wang (2019), knockout of IbGBSSI had no impact on the total starch content in sweet potato tubers. It indicated that sweet potato lines of high-content
amylopectin synthesis have been developed with the knock-out of the gene IbGBSSI by using a highly efficient CRISPR/Cas9 system.

**Table 3 Amylose content (%) of total starch of root tubers**

|  |  |  |  |  |
| --- | --- | --- | --- | --- |
| Line No. | Mass (mg) | OD620 | Content of amylose (%) | |
|  | Mean+SD |
| B23 | spl1 | 10.21 | 0.1970 | 23.5775 | 23.3306+2.4801 |
| spl2 | 9.99 | 0.2050 | 25.6780 |
| spl3 | 9.99 | 0.1800 | 20.7363 |
| 23216004 | spl1 | 10.10 | 0.0790 | 0.7635 | 0.5657 +0.3367 |
| spl2 | 10.10 | 0.0760 | 0.1770 |
| spl3 | 10.19 | 0.0790 | 0.7568 |
| 23216005 | spl1 | 10.26 | 0.0790 | 0.7516 | 0.7613 +0.4862 |
| spl2 | 9.91 | 0.0765 | 0.2800 |
| spl3 | 10.10 | 0.0815 | 1.2523 |

![](https://static.igem.wiki/teams/4887/wiki/images/engineer/20.png)

**Fig. 20 Amylose content (%) of total starch of root tubers**

## **Future work**

Our long-term goal is to develop multiple types of high-performance biodegradable plastics using starch as raw material. Due to the differences in physical and chemical properties
between amylose and amylopectin, different ratios of amylose to amylopectin in raw starch can lead to different properties of starch-based bioplastics. Therefore, by adjusting the ratio
of amylose and amylopectin in raw starch, we can obtain bioplastics with different properties to meet the needs of society and the pure amylose and pure amylopectin will be undoubtedly
the most suitable. However, the existing starch separation and purification technologies usually involve complex processes and expensive equipment. Obtaining high-pure amylose and
high-pure amylopectin with industrial methods will result in high production costs for starch-based bioplastics. It is obviously there is a great demand for natural high-pure amylose and
high-pure amylopectin.

Therefore, in subsequent related research, we will also obtain sweet potato lines with high-amylose synthesis through similar gene editing techniques. Additionally, based on the obtained
sweet potato lines with high-amylopectin synthesis and high-amylose synthesis, we will seek to further enhance the proportion of amylose or amylopectin in these plants through other
biotechnological means so that we can extract the high-pure amylose or high-pure amylopectin directly from sweet potato root tubers. This will eliminate the starch separation and
purification process required for the preparation of starch-based bioplastics, thereby reducing their production cost greatly and promoting the application of starch-based biodegradable
plastics well.

## **Reference**

Wang HX, Wu YL, Zhang YD et al. CRISPR/Cas9-based mutagenesis of starch biosynthetic genes in sweet potato (ipomoea batatas) for the improvement of starch quality. Int. J. Mol. Sci.
2019, 20, 4702; doi:10.3390/ijms20194702

Yang J, Bi HP, Fan WJ et al. Efficient embryogenic suspension culturing and rapid transformation of a range of elite genotypes of sweet potato (Ipomoea batatas [L.] Lam.). Plant. Sci.
2011, 181, 701–711.

Yang J, Moeinzadeh MH, Kuhl H et al. Haplotype-resolved sweet potato genome traces back its hexaploidization history. Nat. Plants 2017, 3, 696–703.

NY/T 2639-2014. Determination of amylose content in rice - Spectrophotometry method

{% endblock %}

{% extends "layout.html" %}
{% block title %}Experiments{% endblock %}
{% block lead %}Describe the research, experiments, and protocols you used in your iGEM project.{% endblock %}
{% block page\_content %}

## What should this page contain?

---

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

If you made Parts this year, please remember to put all information, characterization, and measurement data on the Part's Main Page on the [Registry](http://parts.igem.org/Main_Page).

## Inspirations

---

* [2019 Nantes](https://2019.igem.org/Team%3ANantes/Experiments)
* [2019 TU Eindhoven](https://2019.igem.org/Team%3ATU_Eindhoven/Experiments)
* [2019 Mingdao](https://2019.igem.org/Team%3AMingdao/Demonstrate)
* [2020 Amsterdam](https://2020.igem.org/Team%3AAmsterdam/Experiments)
* [2020 NCTU Formosa](https://2020.igem.org/Team%3ANCTU_Formosa/Experiments)
* [2020 USAFA](https://2020.igem.org/Team%3AUSAFA/Experiments)

{% endblock %}

{% extends "layout.html" %}
{% block title %}Results{% endblock %}
{% block lead %}You can describe the results of your project and your future plans here.{% endblock %}
{% block page\_content %}

## What should this page contain?

---

* Clearly and objectively describe the results of your work.
* Future plans for the project.
* Considerations for replicating the experiments.

## Describe what your results mean

---

* Interpretation of the results obtained during your project. Don't just show a plot/figure/graph/other, tell us what you think the data means. This is an important part of your project that the judges will look for.
* Show data, but remember **all measurement and characterization data must also be on the Part's Main Page on the [Registry](http://parts.igem.org/Main_Page).** Otherwise these data will not be in consideration for any medals or part awards!
* Consider including an analysis summary section to discuss what your results mean. Judges like to read what you think your data means, beyond all the data you have acquired during your project.

## Project Achievements

---

You can also include a list of bullet points (and links) of the successes and failures you have had over your summer. It is a quick reference page for the judges to see what you achieved during your summer.

* A list of linked bullet points of the successful results during your project
* A list of linked bullet points of the unsuccessful results during your project. This is about being scientifically honest. If you worked on an area for a long time with no success, tell us so we know where you put your effort.

## Inspirations

---

* [2019 Newcastle](https://2019.igem.org/Team%3ANewcastle/Results)
* [2019 Munich](https://2019.igem.org/Team%3AMunich/Results)
* [2019 Tec Chihuahua](https://2019.igem.org/Team%3ATec-Chihuahua/Results)
* [2020 Aalto Helsinki](https://2020.igem.org/Team%3AAalto-Helsinki/Results)
* [2020 GreatBay SCIE](https://2020.igem.org/Team%3AGreatBay_SCIE/Results)
* [2020 Queens Canada](https://2020.igem.org/Team%3AQueens_Canada/Results)

{% endblock %}