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.

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