Composite
IBR-T25

Part:BBa_K3254004

Designed by: Yeqing Zong, Qianrui Li   Group: iGEM19_GENAS_China   (2019-10-13)


Int10attB-BsaI site-terminator-Int10attP

We expected that this part can be placed between a promoter and a translational unit part and work as a normally open (NO) switch for the downstreamed gene. and switch to ON state by excising the terminator ECK120020525 between the att sites when it reacted with Int10 integrase ([[Part:BBa_K3254009|BBa_K3254009). Two BsaI restriction site were added between attB site and terminator. As a result, it can work as a normally closed (NC) switch for the gene which was inserted between the two BsaI site and switch to OFF state when it was excised.

Usage and Biology

Visual Result as a Normally Open Switch

  • We conducted a simple test to see if our design met the expection.

Experimental Setup

  • Genetic design principle of the experimental group was described on the page of BBa_K3254012.
  • A P15A-AmpR plasmid was co-transfered into the E.coli DH5α host cell with the reporter plasmid containing this part as the negative control.
  • Single colonies were selected from the experimental LB-agar plate and negative control LB-agar plate, then inoculated into EP tubes with 500 μL M9 supplemented medium containing 500 μM IPTG for overnight growth at 37 °C and 200 rpm.
  • Tubes were centrifuged at 10000g for 1 min. Then observed the GFP fluorescence of the cell precipitations under blue light.
  • 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, 2mM MgSO4, and 100 μM CaCl2.

Results

  • IBR-C35/F55/S37/E21/T25/G22 indicate the experimental systems for phiC31/Int5/Int7/Int8/Int10/TG1 respectively.
  • We observed the GFP fluorescence from the experimental tube as expected.
Visual Results as Normally Open Switches

Quantitative Characterizaion of the Normally Open Switch

Experimental Setup

  • Bacteria harboring the circuits (see the top part of the result image) first inoculated from single colonies into a flat-bottom 96-well plate for overnight growth. Then, the cell cultures were diluted 1000-fold with M9 supplemented medium with 500 μM 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. The arithmetical mean of each sample was determined using FlowJo software.
  • The principle of data processing is shown on the result image.

Results

  • IBR-C35/F55/S37/E21/T25/G22 indicate the experimental systems for phiC31/Int5/Int7/Int8/Int10/TG1 respectively.
  • Compared to other parts, this part performed well.
File:T--GENAS China--primary quantitative characterization.png
Quantitative Characterization of the Normally Open Switches

Thermodynamic Characterization

See the related information on the page of BBa_K3254012.


Orthogonality Characterization

Genetic Design

  • The composition and principle of the experimental system are indicated below.
The composition and principle of the experimental system

Experimental Setup

The reporter plasmid contained this part were co-transferred into E.coli DH5α host with 6 integrase generator plasmids. Then single colonies were inoculated into M9 supplemented medium for overnight growth. Then, the cell cultures were diluted 1000-fold with M9 supplemented medium with 500 μM 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, The cultures were sampled for genotype PCR testing. The principle of genotype identification was shown on the right of results image.

Results

  • IBR-T25 was the plasmid containing this part.
  • The result indicates that this part can only be recombined by int 10 integrase.
  • The sequence (attL or attR) after recombination is agcacgctgataatcagcaagaccaccaacatttcaatcaaggatagtaaaactctcactctt.

File:T--GENAS China--orthogonality test. png


Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI site found at 76
    Illegal BsaI.rc site found at 64


[edit]
Categories
//function/recombination
Parameters
None