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Part:BBa_K1412614:Experience

Designed by: Ruihua Zhang   Group: iGEM14_XMU-China   (2014-10-01)
Revision as of 18:00, 16 October 2014 by D (Talk | contribs) (→‎Activiation)

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Protocol

Verification

Verification.png


Activiation

Transfer 50μL bacterium solution (pLac-RBS (1.0)-CheZ-TT, pLac-RBS (0.01)-CheZ-TT, pLac-RBS (0.3)-CheZ-TT) into 5ml new LB liquid medium

whose chloromycetin concentration is 50ug/ml to culture for 12 hours in 37℃horizontal rotators at 200rpm.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.

Characterization stage

1. Transfer pBAD-RBS-CheZ-TT (BBa_K1412624) into competent cell of E.coli (ΔCheZ) respectively.

2. Coated plates(LB solid medium with the antibiotic concentration 50ug/ml chloromycetin) and culture in 37℃ biochemical incubator for

12h.

3. Select colony to culture in 5ml LB fluid medium, of which the chloromycetin, antibiotic concentration is 50ug/ml.

4. Activation of bacterium: transfer 50uL bacterium medium into a new 5ml LB fluid medium, of which the chloromycetin, antibiotic

concentration is 50ug/ml.

5. Stab of bacterium: We draw three spots on a plate before, then stab 3ul bacterium medium into the M63 semisolid medium at the spots.

6. Culture the bacteria in constant temperature and humidity incubator at 37℃.

Protocol of M63.jpg


Measurement

Xmu project application RBSpromoter02.png

1. 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, … The

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

radius at certain time. Plot the net chemotaxis radius versus time and set the radius of pLac-RBS (1.0)-CheZ-TT

(BBa_K1412000) as RBS strength 1.0. Then we can calculate the RBS efficiency (ΔR2L/ΔR2M) and (R2J/ΔR2M) and compare

them with the official data.


Results

Xmu project application RBSpromoter03.png

Actually, we kept measuring chemotactic diameters of three colonies at different time 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.


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