Difference between revisions of "Part:BBa K3254008"
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The plasmid containing the Int5 expressional unit was co-transfered into E.coli DH5α host with 6 reporter plasmids containing different attP-attB sites sequences. | The plasmid containing the Int5 expressional unit was co-transfered into E.coli DH5α host with 6 reporter plasmids containing different attP-attB sites sequences. | ||
Then single colonies were inoculated into M9 supplemented medium for overnight growth. Then, the cell cultures were diluted 1000-fold with M9 supplemented medium (leakage expression is enough) and growth for another 20 hours. All incubations were carried out using a Digital Thermostatic Shaker maintained at 37 °C and 1000 rpm, using flat-bottom 96-well plates sealed with sealing film. Finally, The cultures were sampled for genotype PCR testing. | Then single colonies were inoculated into M9 supplemented medium for overnight growth. Then, the cell cultures were diluted 1000-fold with M9 supplemented medium (leakage expression is enough) and growth for another 20 hours. All incubations were carried out using a Digital Thermostatic Shaker maintained at 37 °C and 1000 rpm, using flat-bottom 96-well plates sealed with sealing film. Finally, The cultures were sampled for genotype PCR testing. | ||
| − | The principle of genotype identification was shown on the right of results image. | + | The principle of genotype identification was shown on the right of results image. |
| + | *M9 medium (supplemented）: 6.8 g/L Na<sub>2</sub>HPO<sub>4</sub>, 3 g/L KH<sub>2</sub>PO<sub>4</sub>, 0.5 g/L NaCl, 1 g/L NH<sub>4</sub>Cl, 0.34 g/L thiamine, 0.2% casamino acids, 0.4% glucose, 2 mM MgSO<sub>4</sub>, and 100 μM CaCl<sub>2</sub>. | ||
==Results== | ==Results== | ||
Revision as of 08:38, 21 October 2019
Integrase Int8
This is a serine-type phage (LSTP) integrases. The original organism is the Staphylococcus haemolyticus JCSC1435. The product protein of this part can catalyze the recombination between the corresponding attB and attP site (see part BBa_K3254003)
Orthogonality Characterization
We conducted orthogonality tests to see the compatibility between the 6 integrase used in our project. The other integrases include phiC31, Int5, Int7, Int10 and TG1.
Genetic design
The composition and principle of the experimental system are indicated below. More sequence details can be seen on the pages of BBa_K3254011 and BBa_K3254003.
Experimental Setup
The plasmid containing the Int5 expressional unit was co-transfered into E.coli DH5α host with 6 reporter plasmids containing different attP-attB sites sequences. Then single colonies were inoculated into M9 supplemented medium for overnight growth. Then, the cell cultures were diluted 1000-fold with M9 supplemented medium (leakage expression is enough) and growth for another 20 hours. All incubations were carried out using a Digital Thermostatic Shaker maintained at 37 °C and 1000 rpm, using flat-bottom 96-well plates sealed with sealing film. Finally, The cultures were sampled for genotype PCR testing. The principle of genotype identification was shown on the right of results image.
- M9 medium (supplemented）: 6.8 g/L Na2HPO4, 3 g/L KH2PO4, 0.5 g/L NaCl, 1 g/L NH4Cl, 0.34 g/L thiamine, 0.2% casamino acids, 0.4% glucose, 2 mM MgSO4, and 100 μM CaCl2.
Results
The result indicate that Int8 integrase has a good orthogonality with the other 5 att sites and compatible with other integrases. IBR-C35/F55/S37/E21/T25/G22 were the plasmids with phiC31/Int5/Int7/Int8/Int10/TG1 att sites.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 1227
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal AgeI site found at 930
Illegal AgeI site found at 1327 - 1000COMPATIBLE WITH RFC[1000]