Team:HK SSC/Model
Modelling
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 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.. 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 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. .
2. Calibration
We followed iGEM 2019 Plate Reader Abs600 (OD) Calibration protocol, so that we can estimate our number cells.
3. Design and Cloning of BBa_K1894002
Please see the Design page for details.
The cloning methods could be summarized below:
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.