Difference between revisions of "Part:BBa K1412614:Experience"
(→Genetic link stage) |
(→Characterization stage) |
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===='''Characterization stage'''==== | ===='''Characterization stage'''==== | ||
| − | 1.Transfer the part | + | 1.Transfer the part pBAD-RBS-<i>CheZ</i>-TT BBa_K1412624 into <i>E.coli</i> (<i>CheZ</i> knocked out), and coat plates, culture for hours to measure the migration diameter of <i>E.coli</i>. |
| − | measure the migration diameter of <i>E.coli</i>. | + | |
| + | |||
| + | 1. Transfer pBAD-RBS-<i>CheZ</i>-TT<bbpart>BBa_K1412624</bbpart> 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 antibiotic concentration is 50ug/ml chloromycetin. | ||
| + | |||
| + | 4. Activation of bacterium: transfer 50uL bacterium medium into a new 5ml LB fluid medium, of which the antibiotic concentration is | ||
| + | |||
| + | 50ug/ml chloromycetin. | ||
| + | |||
| + | 5. Stab of bacterium: We draw three dots on a plate before, then stab 3ul bacterium medium into the M63 semisolid medium at the dots. | ||
| + | |||
| + | And culture the bacteria in constant temperature and humidity incubator at 37℃. | ||
| + | |||
| + | |||
| + | 6. 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…The chemotaxis rate grows higher over time, larger radius bringing less error. Each radius minus R1 is the net chemotaxis radius at certain time. Plot the net chemotaxis radius versus time and set the radius of pLac-RBS (1.0)-CheZ-TT as RBS strength 1.0. Then we can calculate the RBS efficiency (ΔR2L/ΔR2M) and (R2J/ΔR2M) and compare them with the official data. | ||
===Applications of BBa_K1412614=== | ===Applications of BBa_K1412614=== | ||
Revision as of 04:39, 11 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
Genetic link stage
1.As a skeleton, TT terminator link with target gene CheZ.
2.Then link the skeleton to target gene CheZ-TT.
3.Next, link RBS-CheZ-TT with promoter Plac.
Verification
Characterization stage
1.Transfer the part pBAD-RBS-CheZ-TT BBa_K1412624 into E.coli (CheZ knocked out), and coat plates, culture for hours to measure the migration diameter of E.coli.
1. Transfer pBAD-RBS-CheZ-TTBBa_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 antibiotic concentration is 50ug/ml chloromycetin.
4. Activation of bacterium: transfer 50uL bacterium medium into a new 5ml LB fluid medium, of which the antibiotic concentration is
50ug/ml chloromycetin.
5. Stab of bacterium: We draw three dots on a plate before, then stab 3ul bacterium medium into the M63 semisolid medium at the dots.
And culture the bacteria in constant temperature and humidity incubator at 37℃.
6. 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…The chemotaxis rate grows higher over time, larger radius bringing less error. Each radius minus R1 is the net chemotaxis radius at certain time. Plot the net chemotaxis radius versus time and set the radius of pLac-RBS (1.0)-CheZ-TT as RBS strength 1.0. Then we can calculate the RBS efficiency (ΔR2L/ΔR2M) and (R2J/ΔR2M) and compare them with the official data.
Applications of BBa_K1412614
User Reviews
UNIQ3832bee5717455e7-partinfo-00000001-QINU UNIQ3832bee5717455e7-partinfo-00000002-QINU