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

Revision as of 18:11, 16 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

Activiation

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.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

We draw three dots on a plate before, then stab 3μl bacterium medium into the M63 semisolid medium at the dots.

After that, culture the bacteria in constant temperature and humidity incubator at 37℃.

Measurement

What we need is just a ruler!!!

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.

Finally processing data with excel.

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

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.

Applications of BBa_K1412614

User Reviews

UNIQ7e0a1969bd25eed3-partinfo-00000001-QINU UNIQ7e0a1969bd25eed3-partinfo-00000002-QINU