iGEM 2022 — Team worldshaper-shanghai

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# Notebook

# Lab Notebooks

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# Engineering

# 1.Overview

Anthocyanins takes many physiological functions including antioxidant, antibacterial, anti-aging, antitumor and cardiovascular disease prevention, it was promising in many fields such as food, health care and medicine. To improve the production of anthocyanin in hairy roots of carrot, an inducible anthocyanin biosynthesis system has been successfully developed as the result of series of experiment achievements. Based on the key genes, SbMYB75, SbDEL and XVE, positive vectors of the inducible anthocyanin biosynthesis system (IA) and the negative control for inducible anthocyanin biosynthesis (NC) were constructed using the method of golden gate assembly. Agrobacterium Rhizogenes strains containing vectors IA and NC separately were obtained by electroporation. After infection and co-culture with the Rhizogenes strains, genes of IA and NC were transferred respectively into carrot leaf that formed hairy roots later. GFP expression results showed that successful transformants of the hairy roots ware selected for continual culture in vitro. Anthocyanins were produced in the hairy roots after adding β-estradiol into the liquid media about 30-day culture later. The results showed that different IA carrot hairy root lines had higher relative content of anthocyanins with the range of 28.6525 - 33.6254 relative unites of anthocyanins / g dry weight (RUA/g DW) than that in NC line (1.7044 RUA/g DW). This suggested that IA carrot hairy roots were a potential alternative source for the production of anthocyanins.

# 2. Engineering Success

## 2.1 Gene Cloning

In the beginning, total RNA was isolated from the root of Scutellaria baicalensis using Trizol reagent following the manufacturer’s instructions. The gel electrophoresis results showed that the isolated total RNA had a high enough quality to be used for further molecular investigation. Next, Single-strand cDNAs were synthesized from 5 ug of total RNA and reversely transcribed. The cDNA was used as templates for PCR amplification, SbMYB75-F and SbMYB75-R, SbDEL-F and SbDEL-R, CaMV35S-F and CAMV35S-R were used as PCR primers. The PCR results showed that SbMYB75, SbDEL and CaMV35S were amplified, subcloned and sequenced.

## 2.2 Vector construction

The plasmids of pUC57, pUAP1 and pEC47732 were used as the expression vectors in this project. Plasmid constructions were performed by using the method of golden gate assembly (Carola E. & Sylvestre M., 2014).

### 2.2.1 Level-0 Vector construction

The DNA elements (golden gate assembly, promoters: PAtUBI5, PLexA35S, P2×35S; CDS of Genes: GFP, SbMYB75, SbDEL, XVE; Terminators: Tmas, Thsp18.2, Tnos, T35S) were synthesized by Gensript based on their sequences and were sub-cloned into pUC57. The results showed that 11 Level-0 vectors (pUC57-PAtUBI5, pUC57- PLexA35S, pUC57- P2×35S, pUC57- GFP, pUC57-SbMYB75, pUC57-SbDEL, pUC57-XVE, pUC57-Tmas, pUAP1-Thsp18.2, pUAP1-Tnos, pUAP1-T35S) were constructed.

### 2.2.2 Level-1 Vector construction

Based on the level-0 vectors, the reaction system was shown in Table 1. A PCR reaction was carried out in a thermocycler for 32 cycles of amplification (37℃ for 3 min, 16 ℃ for 4 min) and 32 cycles of amplification (16℃ 15 min；50℃ 5 min，80℃ 5 min). 10 μl of the liagations were used to transform E. coli. Positive clones were confirmed by sequencing.

Table 1 PCR reaction system of level-1 vectors’ construction

| Item | Volume / μL |
| --- | --- |
| Level 1 empty vector (200 ng/μL) | 1.0 |
| promoter | 1.5 |
| CDS | 1.5 |
| terminator | 1.5 |
| NEB T4 Buffer | 1.5 |
| BSA（10X） | 1.5 |
| T4 ligase | 0.5 |
| BsaI | 0.5 |
| ddH2O | 10.0 |
| the whole volume | 20.0 |

The results showed that the four level-1 vectors pEC47732: PAtUBI5- GFP-Tmas (BBa\_K4399008), pEC47742: PLexA35S-SbMYB75- Thsp18.2 (BBa\_K4399009), pEC47751: PLexA35S-SbDEL-Tnos (BBa\_K4399010), pEC47761: P2×35S -XVE-T35S (BBa\_K4399011) were constructed (Figure 1).

Figure 1 The structure of the level 1 vectors

### 2.2.3 Level-2 Vector construction

Based on the level-1 vectors, according to the reaction system shown in Table 2, a PCR reaction ware incubated in a thermocycler for: 37℃ 3 min, 16 ℃ 4 min, 32 cycles; 16℃ 15 min; 50℃ 5 min, 80℃ 5 min. 10 μl of the liagations were used to transform E. coli. Positive clones were confirmed by sequencing and PCR.

Table 2 PCR reaction system of level-2 vectors’ construction

| Item | IA | NC |
| --- | --- | --- |
| Volume/μL | Volume/μL |
| Level 2 empty vector (200 ng/μL) | 1 | 1.0 |
| L1-P1 | 1.5 | 1.5 |
| L1-P2 | 1.5 | 1.5 |
| L1-P3 | 1.5 | 1.5 |
| L1-P4 | 1.5 | - |
| ELE-X | 1.5 | 1.5 |
| NEB T4 Buffer | 1.5 | 1.5 |
| BSA (10X) | 1.5 | 1.5 |
| T4 ligase (NEB) | 0.5 | 0.5 |
| BbsI | 0.5 | 0.5 |
| ddH2O | 7.5 | 9.0 |

For the level 2 vectors, the plasmids pAGM4723 was used and the following vectors IA and NC were constructed (Figure 2)

(1) Vector IA: pAGM4723-PAtUBI5-GFP-Tmas--PLexA35S-SbMYB75-Thsp18.2--PLexA35S-SbDEL-Tnos-- P2×35S -XVE-T35S (BBa\_K4399012)

(2) Vector NC: pAGM4723-PAtUBI5-GFP-Tmas--PLexA35S-SbMYB75-Thsp18.2--PLexA35S-SbDEL-Tnos (BBa\_K4399013)

Figure 2 The structure of the level 2 vectors

Nucleic acid gel electrophoresis detection results showed that the level-2 vector IA and NC showed one band and its molecular weight were greater than 8000 bp (Figure 3). Monoclonal transformation in plates were obtained by E. coli transformation (Figure 4). PCR detection results showed that SbMYB and SbDEL were integrated in monoclones BBa\_K4399012 and BBa\_K4399013 (Figure 5).

Figure 3 Nucleic acid gel electrophoresis results of plasmid detection

Figure 4 The plates of E. coli transformants for vectors IA and NC

Figure 5. Representative PCR analysis for the presence of the SbMYB and SbDAT gene in BBa\_K4399012 and BBa\_K4399013.

A. Representative PCR analysis for the presence of the SbMYB gene in BBa\_K4399012 and BBa\_K4399013.

Line 1-4, PCR results of 4 single colonies of BBa\_K4399012 using SbMYB primers;

Line 5-8, PCR results of 4 single colonies of BBa\_K4399013 using SbMYB primers;

Line 9, positive control;

Line 10, negative control;

Line 11, marker;

B. Representative PCR analysis for the presence of the SbDEL gene in BBa\_K4399012 and BBa\_K4399013.

Line 11-14, PCR results of 4 single colonies of BBa\_K4399012 using SbDEL primers;

Line 15-18, PCR results of 4 single colonies of BBa\_K4399013 using SbDEL primers;

Line 19, positive control;

Line 20, negative control;

Line 21, marker.

## 2.3 Agrobacterium Rhizogenes transformation

The constructed vectors IA and IC were successfully transferred into Agrobacterium Rhizogenes A4 by electroporation. The positive transformants containing both SbMYB75 and SbDEL were selected and cultured on solid TY medium plates containing antibiotics of spectinomycin and kanamycin (Figure 6).

Figure 6 The plates of A. Rhizogenes transformants for vectors IA and NC

## 2.4 Explant infection

Carrot leaves from 4-week-old tissue culture plantlets were used as transformation explants. Carrot leaves were infected by A. Rhizogenes strains containing target genes and co-cultured on the solid MS medium plates containing 50 mM acetosyringone (AS) for 48-72 h at 25°C in the dark and then transferred to MS solid medium containing 400 mg/L cephalexin (cef). 3-5 weeks later after A. Rhizogenes infection, induced hairy roots emerged from the infection site (Figure 6). Hairy roots were derived and cultured in hormone-free half strength MS solid medium. During the culture, some hairy roots turned brown and grown quickly.

Figure 7 Induced hairy root emerged from the infected leaf explants infected by A. Rhizogenes strains containing vectors IA and NC.

The co-transgenic roots were detected by screening for GFP fluorescence signal. With the help of a fluorescence microscope, GFP expression was observed in transgenic hairy roots, successful transformants of hairy roots ware marked and transferred on MS plates (without cefotaxime) in dark at 25 °C for further reproduction.

## 2.5 Hairy root culture and Induction of anthocyanin synthesis

The Carrot hairy roots were cultured in MS liquid medium with 200 mg-L-1 cefotaxime for about 30 days, then 2 μM β-estradiol solution was added in liquid M medium. The results showed that anthocyanin synthesis was successfully induced in the IA hairy roots on the 1st and 5th day, but not induced in the NC hairy roots (Figure 8), implying that the inducible anthocyanin biosynthesis system was successfully constructed.

Figure 8 The anthocyanin synthesis induction result of transgenic carrot hairy roots

## 2.6 Content analysis of anthocyanins

Anthocyanins were extracted with acidified 80% methanol solution. The absorbance of cyanidin 3-O-rutinoside (C3R) at 530 nm and 657 nm were measured in the concentration range of 0.001 - 0.05 mg / mL for the anthocyanin solution (Table 3). The relative content of anthocyanins was calculated with the following formula (Frank M. at al, 2005) (Table 4). The results showed that different carrot hairy root lines produce different anthocyanin contents, the mean relative anthocyanin content in IA hairy root line 16-10 and 16-5 was 28.6525 relative unites of anthocyanins / g dry weight (RUA/g DW) and 33.6254 RUA/g DW, respectively and that in NC line 17-3 was only 1.7044 RUA/g DW.

Table 3 Raw data of anthocyanin content analysis

| Line No. | A530 (nm) | | A657 (nm) | | m(g) | | V (ml) | |
| --- | --- | --- | --- | --- | --- | --- | --- | --- |
| repeat 1 | repeat 2 | repeat 1 | repeat 2 | repeat 1 | repeat 2 | repeat 1 | repeat 2 |
| 16-10-1 | 0.2433 | 0.4108 | 0.0121 | 0.1022 | 0.0102 | 0.0132 | 1.02 | 1.32 |
| 16-10-2 | 0.3051 | 0.3118 | 0.0339 | 0.0484 | 0.0094 | 0.0108 | 0.94 | 1.08 |
| 16-10-3 | 0.2919 | 0.2162 | 0.0186 | 0.0246 | 0.0142 | 0.0107 | 1.42 | 1.07 |
| 16-5-1 | 0.3021 | 0.3182 | 0.0166 | 0.0195 | 0.0108 | 0.0111 | 1.08 | 1.11 |
| 16-5-2 | 0.3530 | 0.2761 | 0.1300 | 0.0134 | 0.0126 | 0.0095 | 1.26 | 0.95 |
| 16-5-3 | 0.4130 | 0.4527 | 0.1155 | 0.0953 | 0.0104 | 0.0111 | 1.04 | 1.11 |
| 16-6-1 | 0.0905 | 0.0904 | 0.1319 | 0.0527 | 0.0153 | 0.0101 | 1.53 | 1.01 |
| 16-6-2 | 0.0647 | 0.0320 | 0.0441 | 0.0057 | 0.0106 | 0.0105 | 1.06 | 1.05 |
| 16-6-3 | 0.0383 | 0.0372 | 0.0060 | 0.0043 | 0.0096 | 0.0094 | 0.96 | 0.94 |
| 17-3-1 | 0.0082 | / | 0.0059 | / | 0.0096 | / | 0.96 | / |
| 17-3-2 | 0.0162 | 0.0487 | 0.0145 | 0.0262 | 0.0107 | 0.0066 | 1.07 | 0.66 |

Table 4 Result of anthocyanin content analysis

| Line No. | relative anthocyanin content (RCA)  relative unites of anthocyanins / g dry weight (RUA/g DW) | | | |
| --- | --- | --- | --- | --- |
|
| repeat 1 | repeat 2 | mean | rmean’ |
| 16-10-1 | 24.0275 | 38.5250 | 31.2763 | 28.6525 |
| 16-10-2 | 29.6625 | 29.9700 | 29.8163 |
| 16-10-3 | 28.7250 | 21.0050 | 24.8650 |
| 16-5-1 | 29.7950 | 31.3325 | 30.5638 | 33.6254 |
| 16-5-2 | 32.0500 | 27.2750 | 29.6625 |
| 16-5-3 | 38.4125 | 42.8875 | 40.6500 |
| 16-6-1 | 5.7525 | 7.7225 | 6.7375 | 4.8654 |
| 16-6-2 | 5.3675 | 3.0575 | 4.2125 |
| 16-6-3 | 3.6800 | 3.6125 | 3.6463 |
| 17-3-1 | 0.67255 | / | 0.6725 | 1.7044 |
| 17-3-2 | 1.2575 | 4.2150 | 2.7363 |

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#### Results Page

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