=== Contribution === Group: HK_SSC, 2019
Author: LEE Hong Kiu, CHOI Justin Yuet Hei

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.
[[File:T--HK_SSC--Gibson.png|500px|thumb|center|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.
{| class="wikitable" |+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. {| class="wikitable" |+E.coli DH5α |- |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 |} {| class="wikitable" |+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 |}
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.
[[File:T--HK_SSC--Equation.jpg|1000px|thumb|center|Figure 4: Gompertz Model]] Curve fitting is done in Matlab.
Coefficients of the Gompertz function when f(x) represents OD₆₀₀ are as follows: {| class="wikitable" |+E.coli DH5α |- | | 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 |} [[File:T--HK_SSC--Ecoli_dh5a.jpg|500px|thumb|center|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
{| class="wikitable" |+E.coli BL21 (DE3) |- | | a | b | c |- | Psb1c3 | 2.776 | 3.352 | 0.6195 |- | BBa_K1894001 | 3.086 | 3.059 | 0.4932 |}
[[File:T--HK_SSC--Ecoli_bl21.png|500px|thumb|center|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:
[[File:T--HK_SSC--BL21NEB.jpg|500px|thumb|center|Figure 6: 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:
{| class="wikitable" |+E.coli DH5α |- | 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 |} We took the logarithm to base 10 value of the above data and conducted curve fitting*. The Gompertz model was used for curve fitting. Here are the results:
{| class="wikitable" |+E.coli DH5α |- | | 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 |} https://www.desmos.com/calculator/k3yyuvk2bt
{| class="wikitable" |+E.coli BL21 (DE3) |- | 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.

===Conclusion=== In E.coli DH5α, the growth rate of shuttle vector BBa_K1894001 transformants is slower than that of Psb1c3 plasmid, but higher than that of PUC19. The final cell yield of BBa_K1894001 transformants was similar to that of pET-Blue 2 transformants. In E.coli BL21 (DE3), the growth rate of BBa_K1894001 shuttle vector was similar to that of Psb1c3 transformants. The final cell yield of E.coli transformed with BBa_K1894001 was even higher than that of BBa_K1894001 transformants. These data shows that the growth rate and the cell yield of E.coli transformed with shuttle vector BBa_K1894001 is comparable to those of common plasmid backbones. This would also mean that standard protocols, including the inoculation time for transformants and transformation, can be used for BBa_K1894001. Users will not have to worry about low cell yields after transformation or insufficient inoculation timing.

We also quantified the number of cell counts in the cell cultures. Cell cultures of BBa_K1894001 in E.coli BL21(DE3) reached 29.3 x 10⁷ cells and BBa_K1894001 in E.coli DH5α 21.4 x 10⁷ cells.
Moreover, we found out that transforming shuttle vector BBa_K1894001 in E.coli BL21(DE3) results in a higher cell yield than in E.coli DH5α.

In conclusion, the large plasmid size of BBa_K1894001 did not adversely affect the E.coli growth rate as expected. Its growth rate is comparable to common vectors including Psb1c3, PUC19 and pET-Blue 2. The results show that BBa_K1894001 is still ideal for cloning. BBa_K1894001 SequenceAndFeatures