Team:HK SSC/Contribution
Summary:
We would like to see how this shuttle vector affects the growth rate of E.coli BL21(DE3) and E.coli DH5α. We will plot a growth curve of transformants of this shuttle vector, and compare to that of transformants of Psb1c3, pET-blue-2 and PUC19.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 common vectors, including Psc1c3, pET-Blue2 and PUC19 have a size less than 4000bp, shuttle vector BBa_K1894001 has a vector size of 6913bp. It is unknown how the large plasmid size or the two ORI will affect the growth rate of E.coli. Data on how this shuttle plasmid affects the growth rate of E.coli has not been provided. If the transformation of this shuttle vector results in low cell yield, protocols may have to be optimized. For example, longer inoculation time will be needed before plasmid purification in order to allow more cells to achieve the ideal DNA yield. We see that previous researchers have also characterized plasmids by measuring the growth rates of transformed cell culturesKarim, Ashty S et al. “Characterization of plasmid burden and copy number in Saccharomyces cerevisiae for optimization of metabolic engineering applications.” FEMS yeast research vol. 13,1 (2013): 107-16. doi:10.1111/1567-1364.12016Klumpp, Stefan. “Growth-rate dependence reveals design principles of plasmid copy number control.” PloS one vol. 6,5 (2011): e20403. doi:10.1371/journal.pone.0020403. Therefore, we decided to add this piece of additional information.Purpose:
The purpose is to find out how this shuttle vector affects the growth rate of E.coli cells by comparing its growth rate to those of other common vectors. If the growth rate is found to be much slower than other vectors, it may not be suitable for cloning. This data will also give the future user an approximate value of the cell yield they should expect.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. Cloning of shuttle vector BBa_K1894001
As shuttle vector BBa_K1894001 is of 6913bp, we could not synthesis it directly. We divided the plasmid into 3 fragments and assembled it using Gibson Assembly. The 3 fragments were designed to have 20-25bp overlap.
Figure 3: The construction of the BBa_K1894001 shuttle vector using Gibson Assembly
Our Sanger sequencing results have shown no undesired mutations in the junctions.4. Transformation
10 ng of plasmid Psc1c3 and shuttle vector BBa_K1894001 were transformed into E.coli BL21(DE3) using iGEM’s transformation protocol. 10ng of plasmid Psc1c3, pET-Blue-2, PUC19 and shuttle vector BBa_K1894001 were transformed into E.coli DH5α using iGEM’s transformation protocol. 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) are compared. E.coli DH5α with inserts Psb1c3 (2070 bp), pET-Blue-2 (3653 bp), PUC19 (2686 bp) and shuttle vector BBa_K1894001 (6913 bp) are compared. The experiments are performed in triplicates.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.Time (h) |
Psb1c3 (OD600) |
PUC19 (OD600) |
pET-Blue 2 (OD600) |
BBa_K1894001 (OD600) |
0 |
0.172 |
0.119 |
0.139 |
0.122 |
0.5 |
0.198 |
0.155 |
0.145 |
0.151 |
1.0 |
0.387 |
0.337 |
0.262 |
0.332 |
1.5 |
0.681 |
0.572 |
0.424 |
0.635 |
2.0 |
1.219 |
0.972 |
0.711 |
0.932 |
2.5 |
1.604 |
1.165 |
0.939 |
1.383 |
3.0 |
2.166 |
1.399 |
1.314 |
1.683 |
3.5 |
2.430 |
1.584 |
1.602 |
1.851 |
4.0 |
2.437 |
1.608 |
1.867 |
1.621 |
4.5 |
2.731 |
1.734 |
2.052 |
2.194 |
16.0 |
3.249 |
1.902 |
2.302 |
2.481 |
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 |
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. (Data not shown)
7. Curve Fitting
Gompertz model is the most frequently used sigmoid model to fit growth data in biology.
Figure 4: Gompertz Model
Curve fitting is done in Matlab. Coefficients of the Gompertz function when f(x) represents OD600 are as follows:
a |
b |
c |
|
Psc1c3 |
3.218 |
4.380 |
0.7485 |
PUC19 |
1.970 |
3.660 |
0.7760 |
pET-Blue 2 |
2.537 |
4.801 |
0.6679 |
BBa_K1894001 |
2.476 |
4.218 |
0.7653 |
Figure 5: Graph of growth curve of E.coli DH5α transformants with plasmids Psb1c3, PUC19, pET-Blue 2, BBa_K1894001
https://www.desmos.com/calculator/tmrmerzvf3?embed
a |
b |
c |
|
Psb1c3 |
2.776 |
3.352 |
0.6195 |
BBa_K1894001 |
3.086 |
3.059 |
0.4932 |
Figure 6: Graph of growth curve of E.coli BL21 (DE3) transformants with plasmids Psb1c3, and BBa_K1894001
https://www.desmos.com/calculator/tmrmerzvf3?embed
Besides, we further compared the growth curve of E.coli DH5α and E.coli BL21(DE3) transformed with BBa_K1894001. The graph is follow:
Figure 7: Graph of growth curve of E.coli DH5α transformants and E.coli BL21 (DE3) transfromants
https://www.desmos.com/calculator/djfz6xqqew?embed
'''Number of Cells'''
We further converted this data into an estimated number of cell count. The data is as follow: Time (h) |
Psb1c3 (cells) |
PUC19 (cells) |
pET-Blue 2 (cells) |
BBa_K1894001(cells) |
0 |
1.60 x 107 |
1.11 x 107 |
1.29 x 107 |
1.14 x 107 |
0.5 |
1.84 x 107 |
1.44 x 107 |
1.35 x 107 |
1.41 x 107 |
1.0 |
3.61 x 107 |
3.14 x 107 |
2.44 x 107 |
3.09 x 107 |
1.5 |
6.35 x 107 |
5.33 x 107 |
3.95 x 107 |
5.92 x 107 |
2.0 |
11.4 x 107 |
9.06 x 107 |
6.63 x 107 |
8.69 x 107 |
2.5 |
15.0 x 107 |
10.9 x 107 |
8.75 x 107 |
12.9 x 107 |
3.0 |
20.2 x 107 |
13.0 x 107 |
12.3 x 107 |
15.7 x 107 |
3.5 |
22.7 x 107 |
14.8 x 107 |
14.9 x 107 |
17.3 x 107 |
4.0 |
22.7 x 107 |
15.0 x 107 |
17.4 x 107 |
1.51 x 107 |
4.5 |
25.5 x 107 |
16.2 x 107 |
19.1 x 107 |
20.5 x 107 |
15.0 |
30.3 x 107 |
17.7 x 107 |
21.4 x 107 |
23.1 x 107 |
a |
b |
c |
|
Psc1c3 |
8.549 |
0.1897 |
0.5358 |
PUC19 |
8.301 |
0.1782 |
0.6291 |
pET-Blue 2 |
8.416 |
0.1807 |
0.4531 |
BBa_K1894001 |
8.425 |
0.1937 |
0.5805 |
Time (h) |
Psb1c3 (cells) |
BBa_K1894001(cells) |
0.0 |
1.00 x 107 |
1.19 x 107 |
0.5 |
1.51 x 107 |
1.66 x 107 |
1.0 |
3.51 x 107 |
3.51 x 107 |
1.5 |
6.31 x 107 |
6.17 x 107 |
2.0 |
11.3 x 107 |
10.6 x 107 |
2.5 |
13.1 x 107 |
12.5 x 107 |
3.0 |
16.3 x 107 |
15.9 x 107 |
3.5 |
17.8 x 107 |
17.0 x 107 |
4.0 |
19.2 x 107 |
17.2 x 107 |
4.5 |
21.1 x 107 |
19.6 x 107 |
15.0 |
26.4 x 107 |
29.3 x 107 |
Possible Errors
We are aware that there may be errors affecting the results. Here are some possible errors that may affect results.1. The cell cultures may not be mixed thoroughly before taking samples out for recording optical density.
2. The cells cultures may be contaminated with other cultures.
3. There may be small objects blocking the emission from the spectrometer.
These may be the possible errors affecting the results. However, we have performed the experiment 3 times in triplicates. We believe that these factors could be minimized by repeating the experiments under the same conditions and taking averages of data.