Difference between revisions of "Part:BBa K1412014:Experience"
| Line 1: | Line 1: | ||
__NOTOC__ | __NOTOC__ | ||
| − | |||
=='''Protocol'''== | =='''Protocol'''== | ||
| Line 9: | Line 8: | ||
[[File:Verification.png |700px]] | [[File:Verification.png |700px]] | ||
| + | |||
'''Figure 1.''' The verification of BBa_K1412014 and BBa_K1412614. | '''Figure 1.''' The verification of BBa_K1412014 and BBa_K1412614. | ||
| + | |||
==='''Activiation'''=== | ==='''Activiation'''=== | ||
| Line 23: | Line 24: | ||
50ug/ml to culture in 37℃ horizontal rotators at 200rpm for 3 hours. | 50ug/ml to culture in 37℃ horizontal rotators at 200rpm for 3 hours. | ||
| + | |||
==='''Culture'''=== | ==='''Culture'''=== | ||
| Line 35: | Line 37: | ||
3. Finally, culture the bacteria in constant temperature and humidity incubator at 37℃ and observe the condition of bacterium growth. | 3. Finally, culture the bacteria in constant temperature and humidity incubator at 37℃ and observe the condition of bacterium growth. | ||
| + | |||
==='''Measurement'''=== | ==='''Measurement'''=== | ||
| Line 48: | Line 51: | ||
colonies from the bottom of the semi-solid medium to avoiding the collapse of semi-solid medium. | colonies from the bottom of the semi-solid medium to avoiding the collapse of semi-solid medium. | ||
| − | 3. Recording data: The diameter of initial colonies(0h) is recorded as R1. And the diameter of the colonies at 12h, 24h, 30h, 36h, 42h... as | + | 3. Recording data: The diameter of initial colonies(0h) is recorded as R1. And the diameter of the colonies at 12h, 24h, 30h, 36h, 42h... |
| + | |||
| + | as R2, R3, R4, R5, R6, R7…. | ||
| − | |||
==='''Results'''=== | ==='''Results'''=== | ||
| Line 58: | Line 62: | ||
[[File:Xmu_project_application_RBSpromoter03.png |400px]] | [[File:Xmu_project_application_RBSpromoter03.png |400px]] | ||
| + | Set the diameter of the colony with promoter Lac as 1.0, and plot the data in excel, We got the following table (Figure 3).The ratio | ||
| − | + | between each colony diameters was fixed after 36 hours. If we set the fixed ratio as relative promoter activities, from our | |
| − | + | ||
| − | each colony diameters was fixed after 36 hours. If we set the fixed ratio as relative promoter activities, from our | + | |
| − | promoter TetR (BBa_R0040) activity is 1.86 relative to promoter Lac (BBa_R0010). Refer to published papers [1] [2], | + | characterization, promoter TetR (BBa_R0040) activity is 1.86 relative to promoter Lac (BBa_R0010). Refer to published papers [1] [2], |
| − | pTetR and pLac has already been measured, and their ratio (pTetR/pLac) is 1.58. So our system is reliable as it | + | promoter activity between pTetR and pLac has already been measured, and their ratio (pTetR/pLac) is 1.58. So our system is reliable as it |
| − | between different promoter activities. However, no published data tell us about the relative promoter activity of | + | could tell the difference between different promoter activities. However, no published data tell us about the relative promoter activity of |
| − | L-arabinose can induce pBAD (BBa_K206000). The characterization of the relative activity of pBAD is carried out | + | pBAD (BBa_K206000), while L-arabinose can induce pBAD (BBa_K206000). The characterization of the relative activity of pBAD is carried out |
| − | arabinose in culture. And the ratio (pBAD/pLac) is 0.37. | + | with 0.02% inducer L-arabinose in culture. And the ratio (pBAD/pLac) is 0.37. |
[[File:Figure_3._The_relative_activity_of_different_promoters-1.jpg |500px]] | [[File:Figure_3._The_relative_activity_of_different_promoters-1.jpg |500px]] | ||
Revision as of 06:52, 17 October 2014
Protocol
Verification
Figure 1. The verification of BBa_K1412014 and BBa_K1412614.
Activiation
1. Transfer 50 μL bacterium solution (pLac-RBS (1.0)-CheZ-TT, pLac-RBS (0.01)-CheZ-TT, pLac-RBS (0.3)-CheZ-TT) into 5 ml new LB liquid
medium whose chloromycetin concentration is 50ug/ml to culture for 12 hours in 37℃ horizontal rotators at 200 rpm.
2. Then transfer another 50μL bacterium solution from the LB above to another new 5ml LB liquid medium whose chloromycetin concentration is
50ug/ml to culture in 37℃ horizontal rotators at 200rpm for 3 hours.
Culture
1. Firstly, draw three dots on a plate before using oily pen from the bottom of culture dish.
2. Secondly, stab 3μl bacterium medium into the M63 semisolid medium at the dots.
3. Finally, culture the bacteria in constant temperature and humidity incubator at 37℃ and observe the condition of bacterium growth.
Measurement
1. Observe the the condition of bacterium growth,and prepare a ruler.
2. When we can see a visible difference of the diameter of semi-solid medium with time pass by, begin to measure the diameter of bacterium
colonies from the bottom of the semi-solid medium to avoiding the collapse of semi-solid medium.
3. Recording data: The diameter of initial colonies(0h) is recorded as R1. And the diameter of the colonies at 12h, 24h, 30h, 36h, 42h...
as R2, R3, R4, R5, R6, R7….
Results
Set the diameter of the colony with promoter Lac as 1.0, and plot the data in excel, We got the following table (Figure 3).The ratio
between each colony diameters was fixed after 36 hours. If we set the fixed ratio as relative promoter activities, from our
characterization, promoter TetR (BBa_R0040) activity is 1.86 relative to promoter Lac (BBa_R0010). Refer to published papers [1] [2],
promoter activity between pTetR and pLac has already been measured, and their ratio (pTetR/pLac) is 1.58. So our system is reliable as it
could tell the difference between different promoter activities. However, no published data tell us about the relative promoter activity of
pBAD (BBa_K206000), while L-arabinose can induce pBAD (BBa_K206000). The characterization of the relative activity of pBAD is carried out
with 0.02% inducer L-arabinose in culture. And the ratio (pBAD/pLac) is 0.37.
Applications of BBa_K1412014
User Reviews
UNIQ9512c292d7421dd2-partinfo-00000000-QINU UNIQ9512c292d7421dd2-partinfo-00000001-QINU