Generator
FlipFlop

Part:BBa_K907005

Designed by: Dong-hui Choe, Soo-in Lee   Group: iGEM12_KAIST_Korea   (2012-09-20)

Dual phase protein generator(mRFP default). mRFP and GFP



<Part Description>
This part is derivative of BBa_K907003, twins with BBa_K907005


KAIST BBa K907005.jpg

KAIST BBa K907005 2.jpg

BBa_E1010(mRFP, reversed) and BBa_0040(wtGFP) are attached to both ends of BBa_K907003 by overlapping extension PCR. This part normally generates mRFP in E.coli(strain MG1655). When BBa_K907000(Bxb1 integrase) inverts the promoter orientation, it starts to generate GFP. When BBa_K907001(Bxb1 excisionase) inverts the promoter orientation back to original state, it expresses mRFP again


<Part Demonstration>


Figure 1. Experimental results of BBa_K907005.

Two ep-tubes designated as BBa_K907005 are containing centrifuged E.coli MG1655 cells possessing BBa_K907005. pTrcHis2A vector containing BBa_K907000, controlled by Trc promoter, transformed into MG1655-BBa_K907005. The double transformed E.coli MG1655 cells showed pink color rather than intense red color of MG1655-BBa_K907005. Gathered cells are not showing perfect yellowish green color because initial state color of mRFP was too strong. With this result, we can say that our designed parts(BBa_K907000, BBa_K907003 and BBa_K907005) are working as we expected. The cells were cultured in 100 mL LB media(1% Cm,AP each) and gathered incubating 12 hours more after induction with 1mM of IPTG. Culture condition was maintained at 37'C and 220 rpm.
Trc promoter, however, has basal level expression of Bxb1 gp35, Mycobacterium Phage Bxb1 DNA integrase, the double transformants were available to change its color without IPTG induction. To diminish the effect of basal transcription level, we performed several optimizations.
Further information are available at KAIST_iGEM_2012 Wiki


Figure 2 & 3. Experimental measurements of intensity of fluorescent.

For numerical value evaluation, cultured cells were sample in every two hours in 96 well plates. The plates were black-colored and flat bottom plates, and measured by plate reader.(TECAN Infinite M200 in Bio Molecular Engineering Laboratory in KAIST, Republic of Korea) The GFPmut3b and mRFP are have maximum excitation wavelength at 501nm and 584nm respectively. Those of emission are 511nm and 607nm. However, the intensity of GFP was measured with excitation wavelength of 501nm and 536nm in emission and 584nm and 619nm in case of mRFP. Because, if the excitation and emission wavelength are too close, the signal interferes and peaks are somewhat unclear.
(Left-A) Induced at 6hr, GFP intensity of double transformant pFlipFlopGFP-pFlipFlopIntergrase(purple)decreased, while the uninduced cells (yellow green) still express GFP. Means that Bxb1 integrase inverts pFlipFlopGFP into RFP state.
(Left-B) Induced at 6hr, the increment of RFP intensity of double transformant pFlipFlopGFP-pFlipFlopIntergrase(purple) is relatively larger than the uninduced group (yellow green). Means that Bxb1 integrase inverts pFlipFlopGFP into RFP state.

(Right-A) Induced at 6hr, the increment of GFP intensity of double transformant pFlipFlopRFP-pFlipFlopIntergrase(purple) is relatively larger than the uninduced group (yellow green). Means that Bxb1 integrase inverts pFlipFlopRFP into GFP state.
(Right-B) Induced at 6hr, the increment of RFP intensity of double transformant pFlipFlopRFP-pFlipFlopIntergrase(purple) is relatively smaller than uninduced group (yellow green). Means that Bxb1 integrase inverts pFlipFlopRFP into GFP state.


Figure 3. Confocal microscopy of pFlipFlopGFPdefault. (A) Uninduced cells, (B) Induced cells


Both (A) and (B) samples carry pFlipFlopGFP and pBxb1 integrase at the same time. They express GFP throughout the culture. However, once sample (B) is induced, Bxb1 integrase inverts the pFlipFlopGFP into its inverted state, thereby starting the expression of mRFP. Compared to sample (A), the expression of mRFP in sample (B) is well observed and it is also clear that the intensity of mRFP grows over time.




<Related Parts>
BBa_K907000 - Mycobacterium Phage Bxb1 integrase
BBa_K907001 - Mycobacterium Phage Bxb1 excisionase
BBa_K907002 - Binary Signal Generator, RBS(reverse) - attB - Promoter - attP - RBS
BBa_K907003 - Binary Signal Generator, Promoter Reversed, RBS(reverse) - attB - Promoter(reverse) - attP - RBS
BBa_K907004 - Dual Phase Protein Generator(GFP default). mRFP and GFP


Sequence and Features

Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 750
    Illegal NheI site found at 773
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal XhoI site found at 838
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 703
    Illegal AgeI site found at 10
    Illegal AgeI site found at 122
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI site found at 723
    Illegal BsaI.rc site found at 806
    Illegal BsaI.rc site found at 1499


[edit]
Categories
Parameters
emission511nm(maximum, BBa_E0040), 607nm(maximum, BBa_E1010), 536nm(for detection, BBa_E0040), 619nm(for detection, BBa_E1010)
excitation501nm(BBa_E0040), 584nm(BBa_E1010)