Difference between revisions of "Part:BBa K1412614:Experience"
(→Measurement) |
|||
| Line 1: | Line 1: | ||
| + | |||
__NOTOC__ | __NOTOC__ | ||
This experience page is provided so that any user may enter their experience using this part.<BR>Please enter | This experience page is provided so that any user may enter their experience using this part.<BR>Please enter | ||
| Line 9: | Line 10: | ||
===='''Genetic link stage'''==== | ===='''Genetic link stage'''==== | ||
| − | 1.As a skeleton, TT terminator link with target gene <i>CheZ</i>. | + | 1. As a skeleton, TT terminator link with target gene <i>CheZ</i>. |
| − | 2.Then link the skeleton to target gene <i>CheZ</i>-TT. | + | 2. Then link the skeleton to target gene <i>CheZ</i>-TT. |
| − | 3.Next, link | + | 3. Next, link RBS-<i>CheZ</i>-TT with promoter Plac. |
====Verification==== | ====Verification==== | ||
| Line 21: | Line 22: | ||
===='''Characterization stage'''==== | ===='''Characterization stage'''==== | ||
| − | 1. Transfer pBAD-RBS-<i>CheZ</i>-TT(<bbpart>BBa_K1412624</bbpart>) into competent cell of <i>E.coli</i>(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 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℃. | |
| − | + | ||
| − | + | ||
| − | + | ||
| − | Culture the bacteria in constant temperature and humidity incubator at 37℃. | + | |
[[File:Protocol_of_M63.jpg |600px]] | [[File:Protocol_of_M63.jpg |600px]] | ||
| − | |||
====Measurement==== | ====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, | + | 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 (<bbpart>BBa_K1412000</bbpart>) as RBS strength 1.0. Then we can calculate the RBS efficiency (ΔR2L/ΔR2M) and (R2J/ΔR2M) and compare them with the official data. |
| − | + | ||
| − | The | + | |
| − | + | ||
| − | certain time. Plot the net chemotaxis radius versus time and set the radius of pLac-RBS (1.0)-CheZ-TT(<bbpart>BBa_K1412000</bbpart>) 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 14:30, 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 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℃.
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, … 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.
Applications of BBa_K1412614
User Reviews
UNIQ8cfbf9205d363a2a-partinfo-00000002-QINU UNIQ8cfbf9205d363a2a-partinfo-00000003-QINU