Part:BBa_K274210:Experience
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Applications of BBa_K274210
Datasheet for Part BBa_K274200, Part BBa_274210 and Part BBa_274220 in E.coli strain MG1655.
For PDF version of this Datasheet: File:Carotene.pdf
Further Characterization of BBa_K274210 in MG1655 and TOP10
Contributed by TEAM SDU-Denmark 2010
Further characterization of the BioBrick K274210 has been performed in Top10 and MG1655 E. coli. The experimental data have shown somewhat similar results for Top10 and MG1655 E. coli.
The experiment was performed as follows:
Over night (ON) cultures were grown from 4 colonies, until the following OD’s were obtained:
Top 10 - no insert OD = 0,008 (100 x diluted)
Top 10 - with K274210 insert OD = 0,011 (100 x diluted)
MG1655 - no insert OD = 0,020 (100 x diluted)
Mg1655 - with K274210 insert OD = 0,017 (100 x diluted)
ON cultures were grown in 110 ml LB media. Colonies with K274210 insert were grown in LB media containing ampicillin. All ON cultures were grown for 20 hours at 37 °C. After 16 hours, 10 ml of the ON cultures were transferred into 110 ml LB media and grown for 4 hours to reach the exponential phase, where the following OD’s were obtained:
Top 10 - no insert OD = 0,049 (100 x diluted)
Top 10 - with K274210 insert OD = 0,044 (100 x diluted)
MG1655 - no insert OD = 0,007 (100 x diluted)
Mg1655 - with K274210 insert OD = 0,009 (100 x diluted)
Cultures with K274210 insert were grown in media containing ampicillin.
100 mL cell culture were centrifuged for 5 min at 14000 RPM. The supernatant was discarded and cells were resuspended in 5 mL acetone (99,9%), except the Top 10 E. coli with the K274210 insert, which was resuspended in 10 mL acetone (source of error). The resuspended cells were sonicated for 5 min. Samples were spun down, the supernatant was transferred to new tubes, and cell debris was discarded. A standard curve was made from pure beta-carotene.
The samples at the OD’s seen above as well as solutions of pure beta-carotene with known concentrations were measured at a fixed wavelength of 456 nm. Known concentrations and their absorbances:
| Concentration | Absorbance |
| 1 mM | 4,000 |
| 100 µM | 2,260 |
| 50 µM | 4,000 |
| 25 µM | 0,155 |
| 10 µM | 0,893 |
| 5 µM | 0,440 |
| 1 µM | 0,075 |
| 100 nM | 0,015 |
| 10 nM | 0,038 |
| 1 nM | 0,005 |
| 100 pM | 0,024 |
The samples and their absorbances:
| Top 10 cells (Absorbance) | MG1655 E. coli mutant (Absorbance) | |
| Stationary phase control | 0,034 | 0,024 |
| Stationary phase with K274210 biobrick insert | 0,319 | 1,549 |
| Expotential phase control | 0,020 | 0,024 |
| Expotential phase with K274210 biobrick insert | 0,034 | 0,033 |
UV-VIS absorbance spectra of the known solutions were obtained, as well as spectra of the samples at the ODs shown above. The spectra of Top 10 and MG1655 in the stationary phase as well as selected spectra of known solutions are shown below:
These results were obtained from one experiment, which will be replicated later for more precision.
Further Characterization of BBa_K274210 in DH5α E.coli
Contributed by British_Columbia 2016 iGEM team.
We expanded on the part characterization by demonstrating β-carotene production in DH5α cells. The cultures were grown in LB media and the carotene was extracted using acetone. The production rate of β-carotene was ~ 3 mg/L calculated using extinction coefficient.
<img src="https://parts.igem.org/File:BC_carotene.png" style="width:500px">
Next we grew DH5α E.coli with BBa_K274210 carotene-producing part transferred to pSB1C3 plasmid and the DH5α control with empty pSB1C3 in M9 minimal media with 0.2% glucose to measure the growth kinatics related to our consortia. </p>
<img src="https://parts.igem.org/File:Growth_ecoli.png" style="width:500px">
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