Difference between revisions of "Part:BBa K1412614:Experience"

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[[File:Xmu_project_application_RBSpromoter02.png |300px]]
 
[[File:Xmu_project_application_RBSpromoter02.png |300px]]
  
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We draw three dots on a plate before, then stab 3μl bacterium medium into the M63 semisolid medium at the dots.  
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1. Firstly, draw three dots on a plate before using  oily pen from the bottom of culture dish.
  
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After that, culture the bacteria in constant temperature and humidity incubator at 37℃.
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2. Secondly, stab 3μl bacterium medium into the M63 semisolid medium at the dots.
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3. Finally, culture the bacteria in constant temperature and humidity incubator at 37℃ and observe the condition of bacterium growth.
  
 
==='''Measurement'''===
 
==='''Measurement'''===

Revision as of 05:38, 17 October 2014

This experience page is provided so that any user may enter their experience using this part.
Please enter how you used this part and how it worked out.

Protocol

Verification

Verification.png

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

Xmu project application RBSpromoter02.png

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

The equipment we need is just a ruler!!!

Ruler.jpg

Use a ruler to measure the diameter of colony from the bottom of the semi-solid medium. The initial colony radius is recorded as R1, and

the radius are measured at 12h, 24h, 30h, 36h, 42h... as R2, R3, R4, R5, R6, R7…and record time and diameter. The migration radius caused

by chemotaxis grows higher over time, larger radius bringing less error. Each radius minus R1 is the net migration. Finally processing data

with excel.

Results

Xmu project application RBSpromoter03.png

Actually, we processing data and set the diameter of the colony with promoter Lac as 1.0. 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_K1412614

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