Difference between revisions of "Part:BBa K3219002"

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Plasmid for in vivo expression of dCas9-sgRNA targeting mcyB
Microcystis Aeruginosa is a noxious cyanobacterium that produces a hepatotoxin, Microcystin. This plasmid silences the McyB gene of Microcystis Aeruginosa UTEX2388 using the dCas9 and sgRNA. According to previous researchers, Microcystin cannot be produced without McyB^[1]

This plasmid can be used for in vivo or in vitro expression of dCas9 and sgRNA.

This plasmid consists of a cyanobacteria shuttle vector, ribosome binding sites, a dCas9-GFP complex and a sgRNA targeting McyB of Microcystis Aeruginosa UTEX 2388 McyB.

Shuttle Vector
The shuttle vector is from team 2016 iGEM team Nanjing_NFLS^[2], part BBa_K1894001. It consists of two origins of replications, f1 ori and pUC ori, which allows it to replicate in both E.coli cells and cyanobacteria cells. It also consists of a t7 promoter, CaMV35S promoter, and Kanamycin resistance gene. The T7 promoter allows expression of the dCas9-GFP-sgRNA in E.coli and the CaMVS promoter allows expression in cyanobacteria.

dCas9
The dCas9, also known as a catalytically dead Cas9 enzyme, is a mutated Cas9 enzyme without endonuclease activity. With the help of a single-guide RNA (sgRNA), it specifically binds to the target sequences and blocks transcript elongation by RNA polymerase. In our project, the dCas9 construct was from the iGEM part registry BBa_K1689013, by teamiGEM15_Peking^[3]. The GFP is added to the C-terminus of the dCas9, connected using a linker. This is to allow visual confirmation of successful transformation and indicates that the dCas9 enzyme has been expressed. There is also a 7x His-Tag, allowing for protein purification.

sgRNA
The sgRNA consists of a handle, a base-pairing region and a terminator. The base-pairing region is designed to target 25 base pairs of the McyB gene of Microcystis Aeruginosa UTEX2388.

Usage

This part can be used by transforming it directly into Microcystis Aeruginosa UTEX 2388 via electroporation or naturual transformation. Our team used the protocols provided by Au Yang Qin ^[4], Nermin Adel El Semary^[5] and Elke Dittmann^[6]. After the expression of dCas9 and sgRNA will result in the repression of the McyB gene. Thus, no Microcystin will be produced.

For in vitro expression, this plasmid ca be transformed to E.coli BL21 (DE3) as it is capable of T7 expression.

Results

1. Successful Transformation
We have successfully transformed our plasmids into Microcystis and spread onto selective plates, showing that the plasmid we constructed is compatible in both E.coli and Microcystis. Besides, our transformed cells demonstrated kanamycin resistance. Living cells are observed on Kanamycin plates as the colonies grew larger each day.

Figure 2: Our transformed Mircocystis cells spread on Kanamycin resistance plate (40x)

2. Plasmid expression We used Microcystin-LR detection kit (WRZ001) from 天河綠洲We believed that our dCas9-sgRNA complex has been successfully been expressed. This is because the Microcystin-LR concentration in our transformed Microcystis was lower than 0.002mg/L, while our control set ups have a higher concentration.

Figure 1: Microcystin detection kit sample

Figure 2: Our results:
1st test (from left): Culture of Microcystis 3 weeks after transformation
2nd test: Water
3rd test: Culture of unsuccessful Microcystis transformation after 3 weeks
4th test: Positive control of Microcystis culture that has not been transformed

Characterization

Introduction

We would like to see how the transformation of this plasmid affects the growth of cells. We transformed our part (BBa_K3219002) into E.coli BL21 and E.coli DH5α, and compared their growth rates to E.coli transformed with common plasmids. In this case, we can see how this large plasmid of 12kbp affects the growth rate of cells, and whether it should be modified in future studies.

Background:

It was found that plasmid sizes and different origin of replications have distinct effects on the growth of E.coli ^[7]. Generally, larger plasmid sizes result in a slower growth rate in E.coli. While our plasmid is larger than 12 kbp, we would like to know whether this large plasmid size will affect the growth rate of E.coli transformed with this plasmid. If the transformation of this plasmid results in a very low cell yield or a slow growth curve, protocols may have to be modified in order to achieve ideal plasmid yield (for conducting plasmid purification) or protein expression.

Methods:

1. Preparation of Cells

E.coli competent cells were prepared using Inoue Method^[8] .

2. Calibration

We followed iGEM 2019 Plate Reader Abs600 (OD) Calibration protocol, s that we can estimate our number cells.

3. Design and Cloning of BBa_K1894002

This plasmid can be divided into 3 parts, the shuttle vector, dCas9-GFP complex and the sgRNA. We assembled the shuttle vector using Gibson Assembly. We cloned the dCas9-GFP construct and the sgRNA into Psb1c3 and pET-Blue respectively. WE assembled the 3 parts using restriction enzyme digestion and ligation.
The cloning methods could be summarized below:

Figure 1: Construction of plasmid BBa_K1894002 (photo from SnapGene)

Our Sanger sequencing results have shown no undesired mutations in the junctions.

4. Transformation

We transformed this plasmid into E.coli DH5α and E.coli BL21 (DE3) using heat shock (42°C for 45 seconds). In order to compare the growth curves of plasmid BBa_K1894002 with other plasmids including part BBa_K1894001, Psb1c3 and pUC19, we also transformed these plasmids into E.coli DH5α and E.coli BL21 (DE3). Transformants were spread onto agar plates with respective antibiotics.

5. Inoculation

Single colonies from each plate were picked. They are inoculated in 3mL of LB with antibiotics for 16 hours at 37°C shaking at 250r.p.m.

6. Measurement

OD600 of the cell cultures were measured and diluted to OD600 ~ 0.1. Then, the diluted culture was inoculated at 37°C shaking at 250r.p.m. OD600 was taken exactly every 30 minutes interval. E.coli BL21 (DE3) with inserts of Psb1c3 (2070) and shuttle vector BBa_K1894001 (6913bp), and plasmid BBa_K1894002 (12280bp) are compared. E.coli DH5α with inserts Psb1c3 (2070 bp), PUC19 (2686 bp), shuttle vector BBa_K1894001 (6913 bp) and plasmid BBa_K1894002 (12280bp) are compared. The experiments are performed in triplicates.

Results

The below graph shows the results of the optical density measurements. The data shown is the average of three replicates.

Conculsion

In DH5α, the growth rate of BBa_K1894002 seems to be distinctively slower than that of other plasmids. The cell yield is also very low, almost half of the cell yield of Psb1c3 transformants. We believe that this result fits our hypothesis that this large plasmid affects cell growth. Protocols have to be optimized when using BBa_K1894002. For example, the inoculation time before plasmid purification can be increased just to achieve the ideal DNA yield, and it may take a longer time for colonies to form after transformation due to the low replication rate.

Sequence and Features

1. ↑ Dittmann, Elke. “Insertional mutagenesis of a peptide synthetase gene that is responsible for hepatotoxin production in the cyanobacterium Microcystis Aeruginosa PCC 7806.” Molecular Microbiology (1997): 779–787. Journal.
2. ↑ https://parts.igem.org/Part:BBa_K1894001
3. ↑ https://parts.igem.org/Part:BBa_K1689013
4. ↑ 章军, 徐虹, 楼士林, 欧阳青. Blue-green alga shuttle plasmid expression vector and method for expressing thymison 'alpha' 1. Thesis. Xia Men: Xia Men University, 1999.
5. ↑ Semary, Nermin Adel El. “Optimized electroporation-induced transformation in Microcystis aeruginosa PCC7806.” Biotechnol. Agron. Soc. Environ (2010): 149-152 . Journal.
6. ↑ Dittmann, Elke. “Insertional mutagenesis of a peptide synthetase gene that is responsible for hepatotoxin production in the cyanobacterium Microcystis Aeruginosa PCC 7806.” Molecular Microbiology (1997): 779–787. Journal.
7. ↑ U. EONG CHEAH, WILLIAM A. WEIGAND, BENJAMIN C. STARK. “Effects of Recombinant Plasmid Size on Cellular Processes in Escherichia coli.” Plasmid (1987): 127-134 . Journal.
8. ↑ Im, H. (2011). The Inoue Method for Preparation and Transformation of Competent E. coli: "Ultra Competent" Cells. Bio-101: e143. DOI: 10.21769/BioProtoc.143.