Part:BBa_K3219002

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

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, part BBa_K1894001. It consists of two origin 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. The GFP is added to the C-terminus of the dCas9, connected using a linker. This is to allow visual confirmation of successful transfromation 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 , Nermin Adel El Semary and Elke Dittmann. 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.

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 . 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 .

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.

Assumptions made:

Assumption	Justification
1. The OD600 = 0.1 is equivalent to 9.3x106	This is according to the calibration curve performed using iGEM standard protocols: Calibration Protocol - Plate Reader Abs600 (OD) Calibration with Microsphere Particles V.2
2. The OD600 of the overnight culture is the maximum OD600.	OD600 remains constant staring from the stationary phase and 16 hours of incubation takes the E.coli to stationary phase .

Results

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

E.coli DH5α

Time (h)	Psb1c3 (OD600)	PUC19 (OD600)	BBa_K1894001 (OD600)	BBa_K1894002 (OD600)
0	0.172	0.119	0.122	0.141
0.5	0.198	0.155	0.151	0.184
1.0	0.387	0.337	0.332	0.283
1.5	0.681	0.572	0.635	0.460
2.0	1.219	0.972	0.932	0.592
2.5	1.604	1.165	1.383	0.765
3.0	2.166	1.399	1.683	0.991
3.5	2.430	1.584	1.851	1.166
4.0	2.437	1.608	1.621	1.369
4.5	2.731	1.734	2.194	1.542
16.0	3.249	1.902	2.481	1.815

E.coli BL21 (DE3)

Time (h)	Psb1c3 (OD600)	BBa_K1894001 (OD600)
0	0.108	0.128
0.5	0.162	0.178
1	0.377	0.377
1.5	0.677	0.662
2	1.213	1.133
2.5	1.406	1.342
3.0	1.746	1.703
3.5	1.911	1.820
4.0	2.063	1.847
4.5	2.266	2.098
16.0	2.836	3.138

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal NheI site found at 3964
  Illegal NheI site found at 12275
  Illegal NotI site found at 777
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BamHI site found at 1685
  Illegal XhoI site found at 716
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 7979
  Illegal NgoMIV site found at 8139
  Illegal NgoMIV site found at 11186
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI site found at 5721
  Illegal SapI site found at 9059