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.
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Further Characterization of BBa_K274210 in MG1655 and TOP10 e. coli has been performed by The 2010 SDU-Denmark iGEM team.
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The experiment was performed as follows:<br>
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Over night (ON) cultures were grown from 4 colonies, until the following OD’s were obtained:
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Top 10 - no insert OD = 0,008 (100 x diluted)<br>
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Top 10 - with K274210 insert OD = 0,011 (100 x diluted)<br>
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MG1655 - no insert OD = 0,020 (100 x diluted)<br>
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Mg1655 - with K274210 insert OD = 0,017 (100 x diluted)<br>
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ON cultures were grown in 110 ml LB media. Colonies with K274210 insert were grown in LB media containing ampicillin.
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All ON cultures were grown for 20 hours at 37 °C.
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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:
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Top 10 - no insert OD = 0,049 (100 x diluted)<br>
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Top 10 - with K274210 insert OD = 0,044 (100 x diluted)<br>
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MG1655 - no insert OD = 0,007 (100 x diluted)<br>
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Mg1655 - with K274210 insert OD = 0,009 (100 x diluted)<br>
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Cultures with K274210 insert were grown in media containing ampicillin.
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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.<br>
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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.
<td>Stationary phase with K274210 biobrick insert</td>
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<td>0,319</td>
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<td>1,549</td>
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</tr>
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<tr>
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<td>Expotential phase control</td>
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<td>0,020</td>
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<td>0,024</td>
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</tr>
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<tr>
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<td>Expotential phase with K274210 biobrick insert</td>
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<td>0,034</td>
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<td>0,033</td>
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</tr>
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</table>
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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:<br>
Further Characterization of BBa_K274210 in MG1655 and TOP10 e. coli has been performed by The 2010 SDU-Denmark iGEM team.
Reference
Hal Alper, et al. Construction of lycopene-overproducing E. coli strains by combining systematic and combinatorial gene knockout targets. Nature Biotechnology 23 (2005).
Nishizaki T, et al. Metabolic engineering of carotenoid biosynthesis in Escherichia coli by ordered gene assembly in Bacillus subtilis. Appl Environ Microbiol. 2007 Feb
Luke Z. Yuan, et al. Chromosomal promoter replacement of the isoprenoid pathway for enhancing carotenoid production in E. coli. Metabolic Engineering 8 (2006).
Luan Tao, et al. Isolation of chromosomal mutations that affect carotenoid production in Escherichia coli: mutations alter copy number of ColE1-type plasmids. FEMS Microbiology Letters 243 (2005)
von Lintig J, et al. Filling the gap in vitamin A research. Molecular identification of an enzyme cleaving beta-carotene to retinal. J Biol Chem. 2000 Apr 21;275(16).
Sequence and Features
Assembly Compatibility:
10
COMPATIBLE WITH RFC[10]
12
COMPATIBLE WITH RFC[12]
21
INCOMPATIBLE WITH RFC[21]
Illegal BamHI site found at 2037
23
COMPATIBLE WITH RFC[23]
25
INCOMPATIBLE WITH RFC[25]
Illegal NgoMIV site found at 1573 Illegal NgoMIV site found at 1703 Illegal AgeI site found at 788
