Difference between revisions of "Part:BBa K1412614:Experience"
(→Characterization stage) |
(→Characterization stage) |
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===='''Characterization stage'''==== | ===='''Characterization stage'''==== | ||
| − | 1. Transfer pBAD-RBS-<i>CheZ</i>-TT<bbpart>BBa_K1412624</bbpart> into competent cell of E.coli (ΔCheZ) respectively. | + | 1. Transfer pBAD-RBS-<i>CheZ</i>-TT(<bbpart>BBa_K1412624</bbpart>) into competent cell of <i>E.coli</i>(ΔCheZ) respectively. |
2. Coated plates(LB solid medium with the antibiotic concentration 50ug/ml chloromycetin) and culture in 37℃ biochemical incubator | 2. Coated plates(LB solid medium with the antibiotic concentration 50ug/ml chloromycetin) and culture in 37℃ biochemical incubator | ||
| Line 29: | Line 29: | ||
3. Select colony to culture in 5ml LB fluid medium, of which the antibiotic concentration is 50ug/ml chloromycetin. | 3. Select colony to culture in 5ml LB fluid medium, of which the antibiotic concentration is 50ug/ml chloromycetin. | ||
| − | 4. Activation of bacterium: | + | 4. Activation of bacterium: transfer 50uL bacterium medium into a new 5ml LB fluid medium, of which the antibiotic concentration is |
50ug/ml chloromycetin. | 50ug/ml chloromycetin. | ||
| Line 35: | Line 35: | ||
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. | 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. | ||
| − | + | Culture the bacteria in constant temperature and humidity incubator at 37℃. | |
| + | |||
[[File:Protocol_of_M63.jpg |600px]] | [[File:Protocol_of_M63.jpg |600px]] | ||
| − | + | ||
| + | ====Measurement==== | ||
| + | |||
| + | 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, R7… | ||
The chemotaxis rate grows higher over time, larger radius bringing less error. Each radius minus R1 is the net chemotaxis radius at | The chemotaxis rate grows higher over time, larger radius bringing less error. Each radius minus R1 is the net chemotaxis radius at | ||
Revision as of 07:51, 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 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 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.
Culture the bacteria in constant temperature and humidity incubator at 37℃.
Measurement
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, 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
UNIQ8c1daf913059ec1f-partinfo-00000001-QINU UNIQ8c1daf913059ec1f-partinfo-00000002-QINU