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.

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)	BBa_K1894002 (OD₆₀₀)
0	0.108	0.128	0.197
0.5	0.162	0.178	0.304
1	0.377	0.377	0.548
1.5	0.677	0.662	0.979
2	1.213	1.133	1.321
2.5	1.406	1.342	1.61
3.0	1.746	1.703	2.031
3.5	1.911	1.820	2.218
4.0	2.063	1.847	2.262
4.5	2.266	2.098	2.391
16.0	2.836	3.138	2.618

We further converted these data to the estimated number of cell counts using the iGEM standard protocols: Calibration Protocol - Plate Reader Abs600 (OD) Calibration with Microsphere Particles V.2.

E.coli DH5α
Time (h)	Psb1c3 (No. of Cells)	PUC19 (No. of Cells)	BBa_K1894001 (No. of Cells)	BBa_K1894002 (No. of Cells)
0	1.83x10⁷	1.11x10⁷	1.14x10⁷	1.31x10⁷
0.5	1.84x10⁷	1.44x10⁷	1.41x10⁷	1.71x10⁷
1.0	3.61x10⁷	3.14x10⁷	3.09x10⁷	2.63x10⁷
1.5	6.35x10⁷	5.33x10⁷	5.92x10⁷	4.29x10⁷
2.0	11.4x10⁷	9.06x10⁷	8.69x10⁷	5.52x10⁷
2.5	15x10⁷	10.9x10⁷	12.9x10⁷	7.13x10⁷
3.0	20.2x10⁷	13.0x10⁷	15.7x10⁷	9.24x10⁷
3.5	22.7x10⁷	14.8x10⁷	17.3x10⁷	10.9x10⁷
4.0	23.9x10⁷	15.0x10⁷	18.4x10⁷	12.8x10⁷
4.5	25.5x10⁷	16.2x10⁷	20.4x10⁷	14.4x10⁷
16.0	30.3x10⁷	17.7x10⁷	23.1x10⁷	16.9x10⁷

E.coli BL21 (DE3)
Time (h)	Psb1c3 (No. of cells)	BBa_K1894001 (No. of cells)	BBa_K1894002 (No. of cells)
0	1x10⁷	1.19x10⁷	1.83x10⁷
0.5	1.51x10⁷	1.66x10⁷	2.83x10⁷
1	3.51x10⁷	3.51x10⁷	5.10x10⁷
1.5	6.31x10⁷	6.17x10⁷	9.13x10⁷
2	11.3x10⁷	10.6x10⁷	12.3x10⁷
2.5	13.1x10⁷	12.5x10⁷	15.0x10⁷
3.0	16.3x10⁷	15.9x10⁷	18.9x10⁷
3.5	17.8x10⁷	17.0x10⁷	20.5x10⁷
4.0	19.2x10⁷	18.1x10⁷	21.4x10⁷
4.5	21.1x10⁷	19.6x10⁷	22.3x10⁷
16.0	26.4x10⁷	29.3x10⁷	24.4x10⁷

7. Curve Fitting

Gompertz model is the most frequently used sigmoid model to fit growth data in biology.

We took the logarithm to base 10 value, and plotted a growth curve using the Grompertz model. Here are the results:

Equation of BBa_K3219002 transformed in E.coli

Growth curve of E.coli DH5α Transfromants

Link to graph.

Equation of BBa_K3219002 transformed to E.coli BL21 (DE3)

Growth curve of E.coli BL21 (DE3) Transfromants

Link to graph.

https://static.igem.org/mediawiki/2019/9/95/T--HK_SSC--Our_part_dh5a.png

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.

References

↑ 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.

↑ http://parts.igem.org/Part:BBa_K1894001

↑ http://parts.igem.org/Part:BBa_K1689013

↑ 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.

↑ Semary, Nermin Adel El. “Optimized electroporation-induced transformation in Microcystis aeruginosa PCC7806.” Biotechnol. Agron. Soc. Environ (2010): 149-152 . Journal.