Difference between revisions of "Part:BBa K419012:Experience"
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===Applications of BBa_K419012=== | ===Applications of BBa_K419012=== | ||
| + | '''The Part BBa_k419012 (constitutive promoter-CrtEBIY)Verification of the Part BBa_k419012''' | ||
| + | |||
| + | We use constitutive promoter to drive the CrtEBIY composite for beta-carotene synthesis. The CrtEBIY composite (BBa_K274210) was obtained from the distribution part of iGEM 2009 Cambridge and digested with restriction enzymes, EcoR I and Pst I, then ligated to EcoR I and Pst I sites of pSB1C3, the iGEM 2010 standard backbone. We verified our composite for synthesis system by EcoRI and PstI digestion and the agarose gel electrophoresis result was shown. The arrow (red) indicates the CrtEBIY with constitutive promoter (R001) DNA fragment. The synthesis system composite was transformed into DH5-alpha E. coli for further experiment. | ||
| + | |||
| + | [[Image:photo2.jpg]] | ||
| + | |||
| + | ===Demonstration of Synthesis System [contains the part BBa_k419012 (constitutive promoter-CrtEBIY) in the iGEM 2010 standard backbone pSB1C3]=== | ||
| + | '''Growth Curve of Synthesis System E. coli ''' | ||
| + | |||
| + | To verify the beta-carotene production by our synthesis system E. coli (DH5), which contains the part BBa_k419012 (beta-carotene producing genes CrtEBIY with constitutive promoter) in the iGEM 2010 standard backbone pSB1C3, the bacterial culture of our synthesis system was performed and the growth curve was determined in the experiment. | ||
| + | Single colony of our synthesis system or control (without beta-carotene genes) bacteria was inoculated into LB medium containing 34 g/ml chloramphenical , then incubated at 37℃ overnight. The next day, the absorbance of the overnight cultures was measured at 600 nm and 0.1 OD of diluted bacterial culture (1 ml) was inoculated into 40 ml fresh LB/chloramphenical broth, then grow the culture at 37℃ incubator. We aliquot the bacterial culture (1 ml) every 2 hours and measured the bacterial growth by spectrophotometer at 600nm. Finally, we determine and draw the bacterial growth curve of our synthesis system E. coli. The Figure shows the growth curve of our synthesis system or control E. coli. The result shows that both control (green curve) and synthesis system (synthesizer, orange curve) E. coli reached to plateau at 8 h and grown equally well. We prove that the growth of synthesis system (containing part BBa_k274200) is well in the same growth condition compared with control. | ||
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| + | [[Image:photo3.jpg]] | ||
| + | |||
| + | ===Beta-carotene Productivity of Synthesis System E. coli=== | ||
| + | |||
| + | '''The production curve of beta-carotene in synthesis system E. coli''' | ||
| + | The level of beta-carotene production was measured every two hours. The beta-carotene produced by the synthesis system E. coli (1 ml) was extracted by acetone, then the absorbance was determined at 450nm by spectrophotometer. Figure shows the absorbance of beta-carotene. The result shows that that the level of the beta-carotene in our synthesis system contained part BBa_K419012 (synthesizer, orange curve) was elevated at 4 hour and reached a maximum level at 8 h. No beta-carotene was produced in the control E. coli (green curve). | ||
| + | |||
| + | [[Image:photo4.jpg]] | ||
| + | |||
| + | The level of beta-carotene was calculated by the concentration standard curve of beta-carotene. The commercial available beta-carotene was purchased and established the concentration curve of beta-carotene as shown in the following Figure. | ||
| + | |||
| + | [[Image:photo5.jpg]] | ||
| + | |||
| + | '''Estimation of the bacterial numbers of synthesis system E. coli''' | ||
| + | |||
| + | Figure shows that the calculation of the total number of the synthesis system E. coli. The bacterial cultures were series diluted and plated on LB agar contained cploramphenical. As shown in figure, we count the number of colony at 10^6x and 10^7x dilution of 6h bacterial culture. We calculate the average bacterial numbers at 6 h culture is 7x10^8. | ||
| + | |||
| + | [[Image:photo6.jpg]] | ||
| + | |||
| + | '''The productivity of beta-carotene produced by the synthesis system E. coli''' | ||
| + | |||
| + | Based on the standard concentration curve of beta-carotene as mention above, we calculate the productivity of beta-carotene produced by our synthesis system E. coli. | ||
| + | |||
| + | y= 0.1865x + 0.0014 | ||
| + | y: absorbance at 450 nm, x: concentration of beta-carotene (microg) | ||
| + | |||
| + | For example, we choose two time point to measure the productivity of beta-carotene | ||
| + | At 6 h culture, y= 0.204 x1 = 1.086 (microg) | ||
| + | At 8 h culture, y= 0.248 x2 = 1.322 (microg) | ||
| + | |||
| + | At 6 h culture, total bacterial numbers = 7x10^8 | ||
| + | Productivity of beta-carotene per cell | ||
| + | 1.086 / 7 x10^8 = 1.551 x10^-9 | ||
| + | At 8 h culture, total bacterial numbers = 1.018x10^9 | ||
| + | Productivity of beta-carotene per cell | ||
| + | 1.322 / 1.018x10^9 = 1.299 x10^-9 | ||
| + | |||
| + | Average productivity per cell: | ||
| + | (1.551 x10^-9 + 1.299 x10^-9) / 2 = 1.425 x10^-9 (microg) [0.001425 pg] | ||
| + | |||
| + | The aliquot of 8 h culture (1 ml) was used to determine the concentration of beta-carotene. The maximum productivity of beta-carotene is estimated approximately 1.322 mg/L. | ||
===User Reviews=== | ===User Reviews=== | ||
Latest revision as of 13:37, 6 November 2010
Applications of BBa_K419012
The Part BBa_k419012 (constitutive promoter-CrtEBIY)Verification of the Part BBa_k419012
We use constitutive promoter to drive the CrtEBIY composite for beta-carotene synthesis. The CrtEBIY composite (BBa_K274210) was obtained from the distribution part of iGEM 2009 Cambridge and digested with restriction enzymes, EcoR I and Pst I, then ligated to EcoR I and Pst I sites of pSB1C3, the iGEM 2010 standard backbone. We verified our composite for synthesis system by EcoRI and PstI digestion and the agarose gel electrophoresis result was shown. The arrow (red) indicates the CrtEBIY with constitutive promoter (R001) DNA fragment. The synthesis system composite was transformed into DH5-alpha E. coli for further experiment.
Demonstration of Synthesis System [contains the part BBa_k419012 (constitutive promoter-CrtEBIY) in the iGEM 2010 standard backbone pSB1C3]
Growth Curve of Synthesis System E. coli
To verify the beta-carotene production by our synthesis system E. coli (DH5), which contains the part BBa_k419012 (beta-carotene producing genes CrtEBIY with constitutive promoter) in the iGEM 2010 standard backbone pSB1C3, the bacterial culture of our synthesis system was performed and the growth curve was determined in the experiment. Single colony of our synthesis system or control (without beta-carotene genes) bacteria was inoculated into LB medium containing 34 g/ml chloramphenical , then incubated at 37℃ overnight. The next day, the absorbance of the overnight cultures was measured at 600 nm and 0.1 OD of diluted bacterial culture (1 ml) was inoculated into 40 ml fresh LB/chloramphenical broth, then grow the culture at 37℃ incubator. We aliquot the bacterial culture (1 ml) every 2 hours and measured the bacterial growth by spectrophotometer at 600nm. Finally, we determine and draw the bacterial growth curve of our synthesis system E. coli. The Figure shows the growth curve of our synthesis system or control E. coli. The result shows that both control (green curve) and synthesis system (synthesizer, orange curve) E. coli reached to plateau at 8 h and grown equally well. We prove that the growth of synthesis system (containing part BBa_k274200) is well in the same growth condition compared with control.
Beta-carotene Productivity of Synthesis System E. coli
The production curve of beta-carotene in synthesis system E. coli The level of beta-carotene production was measured every two hours. The beta-carotene produced by the synthesis system E. coli (1 ml) was extracted by acetone, then the absorbance was determined at 450nm by spectrophotometer. Figure shows the absorbance of beta-carotene. The result shows that that the level of the beta-carotene in our synthesis system contained part BBa_K419012 (synthesizer, orange curve) was elevated at 4 hour and reached a maximum level at 8 h. No beta-carotene was produced in the control E. coli (green curve).
The level of beta-carotene was calculated by the concentration standard curve of beta-carotene. The commercial available beta-carotene was purchased and established the concentration curve of beta-carotene as shown in the following Figure.
Estimation of the bacterial numbers of synthesis system E. coli
Figure shows that the calculation of the total number of the synthesis system E. coli. The bacterial cultures were series diluted and plated on LB agar contained cploramphenical. As shown in figure, we count the number of colony at 10^6x and 10^7x dilution of 6h bacterial culture. We calculate the average bacterial numbers at 6 h culture is 7x10^8.
The productivity of beta-carotene produced by the synthesis system E. coli
Based on the standard concentration curve of beta-carotene as mention above, we calculate the productivity of beta-carotene produced by our synthesis system E. coli.
y= 0.1865x + 0.0014
y: absorbance at 450 nm, x: concentration of beta-carotene (microg)
For example, we choose two time point to measure the productivity of beta-carotene At 6 h culture, y= 0.204 x1 = 1.086 (microg) At 8 h culture, y= 0.248 x2 = 1.322 (microg)
At 6 h culture, total bacterial numbers = 7x10^8
Productivity of beta-carotene per cell 1.086 / 7 x10^8 = 1.551 x10^-9 At 8 h culture, total bacterial numbers = 1.018x10^9 Productivity of beta-carotene per cell 1.322 / 1.018x10^9 = 1.299 x10^-9
Average productivity per cell: (1.551 x10^-9 + 1.299 x10^-9) / 2 = 1.425 x10^-9 (microg) [0.001425 pg]
The aliquot of 8 h culture (1 ml) was used to determine the concentration of beta-carotene. The maximum productivity of beta-carotene is estimated approximately 1.322 mg/L.
User Reviews
UNIQ7d0cce84b82aeb8b-partinfo-00000000-QINU UNIQ7d0cce84b82aeb8b-partinfo-00000001-QINU