Difference between revisions of "Part:BBa K3254009"

 
 
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This is a serine-type phage (LSTP) integrases. The original organism is the Streptococcus pneumoniae 670-6B. The product protein of this part can catalyze the recombination between the corresponding attB and attP site (see part [[Part:BBa_K3254004|BBa_K3254004]]).  
 
This is a serine-type phage (LSTP) integrases. The original organism is the Streptococcus pneumoniae 670-6B. The product protein of this part can catalyze the recombination between the corresponding attB and attP site (see part [[Part:BBa_K3254004|BBa_K3254004]]).  
 +
 +
=Thermodynamic Characterization=
 +
*We characterized the thermodynamic characteristic and orthogonality between Int10 and 5 other LSTP.
 +
*A new translational unit [[Part:BBa_K3254012|BBa_K3254012]] including this part was constructed.
 +
*The cis-element used in this experiments were [[Part:BBa_K3254004|BBa_K3254004]] which contains the Int10 att sites.
 +
*We constructed an IPTG inducible system to control the expression level by the IPTG concentration in E.coli DH5α host.
 +
*The distribution ratios of the cells with original att sites or recombined att sites were measured using a flow cytometry.
 +
 +
==Genetic design==
 +
*The composition and principle of the experimental system are indicated below. More details can be seen on the pages of [[Part:BBa_K3254012|BBa_K3254012]] and [[Part:BBa_K3254004|BBa_K3254004]].
 +
*[[Part:BBa_K3254025|BBa_K3254025]] can be seen as a reference of device structure.
 +
 +
[[File:T--GENAS_China--excision_with_backbone.PNG|200px|thumb|left|The composition and principle of the experimental system]]
 +
 +
==Experimental Setup==
 +
The two plasmids were co-transformed into E.coli DH5α host.
 +
Then we selected colonies with no observable fluoresce and inoculated into M9 supplemented medium for overnight growth. Then, the cell cultures were diluted 1000-fold with M9 supplemented medium with gradient concentrations of  IPTG inducer 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, 3-μL samples each culture were transferred to a new 96-well plate containing 200 μL per well of PBS supplemented with 2 mg/mL kanamycin. The fluorescence distribution of each sample was assayed using a flow cytometry.
 +
*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==
 +
*The recombination rate could be controlled by IPTG concentration and has a narrow hypersensitivity response interval (the orange circle indicate the Int10 testing group).
 +
*Because the Ptac promoter has a nearly-linear response curve (see [[Part:BBa_K3254025|BBa_K3254025]]), it is more likely that the hypersensitivity is due to the characteristics of the integrase itself rather than the characteristics of the transcriptional regulation system.
 +
 +
[[File:T--GENAS_China--RR-IPTG.png]]
 +
 +
=Orthogonality Characterization=
 +
We conducted orthogonality tests to see the compatibility between the 6 integrase used in our project. The other integrases include Int5, Int7, Int8, Int10 and TG1.
 +
 +
==Genetic Design==
 +
Similar to the design of thermodynamic characterization. Each integrase generator plasmid were co-transferred with 6 plasmid containing different sets of cis-elements.
 +
 +
==Experimental Setup==
 +
The growth condition is similar to the thermodynamic characterization but every sample were full induced with 500 μM IPTG. Then we used specific primers to identify the genotype of att sites by PCR. The principle of genotype identification was shown on the right of result image.
 +
 +
==Results==
 +
The results indicate that Int10 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.
 +
 +
[[File:T--GENAS_China--orthogonality_test.png]]
 +
  
 
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<span class='h3bb'>Sequence and Features</span>
 
<span class='h3bb'>Sequence and Features</span>
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<partinfo>BBa_K3254009 Sequence And Features</partinfo>
  
  

Latest revision as of 12:56, 21 October 2019


Integrase Int10

This is a serine-type phage (LSTP) integrases. The original organism is the Streptococcus pneumoniae 670-6B. The product protein of this part can catalyze the recombination between the corresponding attB and attP site (see part BBa_K3254004).

Thermodynamic Characterization

  • We characterized the thermodynamic characteristic and orthogonality between Int10 and 5 other LSTP.
  • A new translational unit BBa_K3254012 including this part was constructed.
  • The cis-element used in this experiments were BBa_K3254004 which contains the Int10 att sites.
  • We constructed an IPTG inducible system to control the expression level by the IPTG concentration in E.coli DH5α host.
  • The distribution ratios of the cells with original att sites or recombined att sites were measured using a flow cytometry.

Genetic design

  • The composition and principle of the experimental system are indicated below. More details can be seen on the pages of BBa_K3254012 and BBa_K3254004.
  • BBa_K3254025 can be seen as a reference of device structure.
The composition and principle of the experimental system

Experimental Setup

The two plasmids were co-transformed into E.coli DH5α host. Then we selected colonies with no observable fluoresce and inoculated into M9 supplemented medium for overnight growth. Then, the cell cultures were diluted 1000-fold with M9 supplemented medium with gradient concentrations of IPTG inducer 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, 3-μL samples each culture were transferred to a new 96-well plate containing 200 μL per well of PBS supplemented with 2 mg/mL kanamycin. The fluorescence distribution of each sample was assayed using a flow cytometry.

  • 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 recombination rate could be controlled by IPTG concentration and has a narrow hypersensitivity response interval (the orange circle indicate the Int10 testing group).
  • Because the Ptac promoter has a nearly-linear response curve (see BBa_K3254025), it is more likely that the hypersensitivity is due to the characteristics of the integrase itself rather than the characteristics of the transcriptional regulation system.

T--GENAS China--RR-IPTG.png

Orthogonality Characterization

We conducted orthogonality tests to see the compatibility between the 6 integrase used in our project. The other integrases include Int5, Int7, Int8, Int10 and TG1.

Genetic Design

Similar to the design of thermodynamic characterization. Each integrase generator plasmid were co-transferred with 6 plasmid containing different sets of cis-elements.

Experimental Setup

The growth condition is similar to the thermodynamic characterization but every sample were full induced with 500 μM IPTG. Then we used specific primers to identify the genotype of att sites by PCR. The principle of genotype identification was shown on the right of result image.

Results

The results indicate that Int10 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.

T--GENAS China--orthogonality test.png


Sequence and Features BBa_K3254009 Sequence And Features Not understood