Composite

Part:BBa_K1937010:Design

Designed by: Oumnia KARIM   Group: iGEM16_Toulouse_France   (2016-10-14)
Revision as of 18:47, 17 October 2016 by Brice (Talk | contribs)

Part: BBa_K1937010 (pSBBS0K-Epsilon/MazF)
(Chassis Bacillus subtilis, carrier plasmid pSBBS0K-mini)
Length: 6855 bp

Background: This Epsilon/MazF part is one of the two components of a double toxin-antitoxin system. The gene Epsilon codes for a toxin countered by the anti-toxin Zeta (Zielenkiewicz and Cegłowski, 2005 ; Mutschler et al., 2011). The gene MazF encodes the anti-toxin for the MazE toxin (Bravo et al., 1987, Zhang et al., 2005, Wang et al., 2013). The part was designed to be carried on a plasmid while its counterpart MazE/Zeta was carried on another. Unfortunately, only Epsilon/MazF part was successfully cloned in E. coli. This BioBrick is a part developed by the Toulouse 2016 iGEM team and cloned on the pSBBS0K-mini plasmid BBa_K1937001 (http://2016.igem.org/Team:Toulouse_France)


This part: Epsilon and MazF are under the control of the promoter Pveg (constitutive promotor for B. subtilis) followed by a strong RBS. A theophylline-dependent riboswitch was used to prevent the MazE toxin expression during the cloning steps in presence of theophylline (Topp and Gallivan, 2008). Indeed, with an expressed toxin, it would be impossible to clone the Biobrick in E. coli. SacII and KpnI are the restriction sites placed respectively before and after the riboswitch so that this fragment can easily be replaced depending on the control we want to apply to the bacteria. The construction was created by integrating the EcorI-PstI fragment from BBa_K1937009 in the pSBBS0K-mini plasmid (BBa_K1937001).


BBa K1937007-map1.png

Validation: The antifungal property was tested against Talaromyces funiculosus. Briefly, plates were evenly inoculated with the fungus and paper patches were added with either copper sulfate (positive control), water (negative control), Bacillus subtilis with pSBBS0K-mini (negative control, BBa_K1937001) or Bacillus subtilis with pSBBS0K-mini-AF-A.

Igemtoulouse2016af.jpg


Light but reproducible inhibition halo were observed after 3 days and still present after 8 days only when the Bacillus subtilis strain carry the antifungal plasmid.


Sequence:



Map
BBa K1937008-genemap.png

gAATTCGCGGCCGCTTCTAGAGggagttctgagaattggtatgccttataagtccaattaacagttgaaaacctgcataggagagctatgcgggttttttattttacataatgatacataatttaccgaaacttgcggaacataattgaggaatcatagaattttgtcaaaataattttattgacaacgtcttattaacgttgatataatttaaattttatttgacaaaaatgggctcgtgttgtacaataaatgtagtaaaggtggtgaaGCTAGCATGTTTGCTAAGAGATTCAAAACATCCTTACTCCCTTTGTTCGCGGGATTTTTGCTGCTGTTTCACCTCGTACTGGCCGGTCCGGCAGCAGCCAGTGCAGAAACCGCGAATAAGTCTAATGAGCATAGACATCAAGGCCCGATTTTTGATACAAGACCGTCACCGTTTAATCCGAATCAACCGAGACCGGGCCCGATTTATaaaggtggtgaaATGTTCGCTAAAAGATTCAAAACCAGCCTGCTGCCTTTATTTGCCGGATTCCTCCTTCTGTTCCACTTAGTGCTCGCGGGTCCGGCAGCAGCAAGCGCCGAAACGGCAAATAAGTCAAATGAGTCAAGTTACGGGCGAAAATCAAAGTACGGCTGCGGGCGAAGATCAAGCTATGACCAGGCACCGCGGGTCGACtgTTATCCGGCTATATTGCAGGAGCGATTATGAAACAAGAAGTTATCCTGGTACTCGACTGTGGCGCGACCAATGTCAGGGCCATCGCGGTTAATCGGCAGGGCAAAATTGTTGCCCGCGCCTCAACGCCTAATGCCAGCGATATCGCGATGGAAAACAACACCTGGCACCAGTGGTCTTTAGACGCCATTTTGCAACGCTTTGCTGATTGCTGTCGGCAAATCAATAGTGAACTGACTGAATGCCACATCCGaGGTATCGCCGTCACCACCTTTGGTGTGGATGGCGCTCTGGTAGATATACTAGTAGCGGCCGCTGCAGGCGCAACGCAATTAATGTGAGTTAGGATCCTGCCTCGCGCGTTTCGGTGATGACGGTGAAAACCTCTGACACATGCAGCTCCCGGAGACGGTCACAGCTTGTCTGTAAGCGGATGCCGGGAGCAGACAAGCCCGTCAGGGCGCGTCAGCGGGTGTTGGCGGGTGTCGGGGCGCAGCCATGACCCAGTCACGTAGCGATAGCGGAGTGTATACTGGCTTAACTATGCGGCATCAGAGCAGATTGTACTGAGAGTGCACCATATGCGGTGTGAAATACCGCACAGATGCGTAAGGAGAAAATACCGCATCAGGCGCTCTTCCGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAAGGCGGTAATACGGTTATCCACAGAATCAGGGGATAACGCAGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCATAGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGGACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATTATCAAAAAGGATCTTCACCTAGATCCTTTTAAATTAAAAATGAAGTTTTAAATCAATCTAAAGTATATATGAGTAAACTTGGTCTGACAGTTACCAATGCTTAATCAGTGAGGCACCTATCTCAGCGATCTGTCTATTTCGTTCATCCATAGTTGCCTGACTCCCCGTCGTGTAGATAACTACGATACGGGAGGGCTTACCATCTGGCCCCAGTGCTGCAATGATACCGCGAGACCCACGCTCACCGGCTCCAGATTTATCAGCAATAAACCAGCCAGCCGGAAGGGCCGAGCGCAGAAGTGGTCCTGCAACTTTATCCGCCTCCATCCAGTCTATTAATTGTTGCCGGGAAGCTAGAGTAAGTAGTTCGCCAGTTAATAGTTTGCGCAACGTTGTTGCCATTGCTGCCGGCATCGTGGTGTCACGCTCGTCGTTTGGTATGGCTTCATTCAGCTCCGGTTCCCAACGATCAAGGCGAGTTACATGATCCCCCATGTTGTGCAAAAAAGCGGTTAGCTCCTTCGGTCCTCCGATCGTTGTCAGAAGTAAGTTGGCCGCAGTGTTATCACTCATGGTTATGGCAGCACTGCATAATTCTCTTACTGTCATGCCATCCGTAAGATGCTTTTCTGTGACTGGTGAGTACTCAACCAAGTCATTCTGAGAATAGTGTATGCGGCGACCGAGTTGCTCTTGCCCGGCGTCAACACGGGATAATACCGCGCCACATAGCAGAACTTTAAAAGTGCTCATCATTGGAAAACGTTCTTCGGGGCGAAAACTCTCAAGGATCTTACCGCTGTTGAGATCCAGTTCGATGTAACCCACTCGTGCACCCAACTGATCTTCAGCATCTTTTACTTTCACCAGCGTTTCTGGGTGAGCAAAAACAGGAAGGCAAAATGCCGCAAAAAAGGGAATAAGGGCGACACGGAAATGTTGAATACTCATACTCTTCCTTTTTCAATATTATTGAAGCATTTATCAGGGTTATTGTCTCATGAGCGGATACATATTTGAATGTATTTAGAAAAATAAACAAATAGGGGTTCCGCGCACATTTCCCCGAAAAGTGCCACCTGACGTCTAAGAAACCATTATTATCATGACATTAACCTATAAAAATAGGCGTATCACGAGGCCCTTTCGTCTTCAAGAGTTCCTTAAGGAACGTACAGACGGCTTAAAAGCCTTTAAAAACGTTTTTAAGGGGTTTGTAGACAAGGTAAAGGATAAAACAGCACAATTCCAAGAAAAACACGATTTAGAACCTAAAAAGAACGAATTTGAACTAACTCATAACCGAGAGGTAAAAAAAGAACGAAGTCGAGATCAGGGAATGAGTTTATAAAATAAAAAAAGCACCTGAAAAGGTGTCTTTTTTTGATGGTTTTGAACTTGTTCTTTCTTATCTTGATACATATAGAAATAACGTCATTTTTATTTTAGTTGCTGAAAGGTGCGTTGAAGTGTTGGTATGTATGTGTTTTAAAGTATTGAAAACCCTTAAAATTGGTTGCACAGAAAAACCCCATCTGTTAAAGTTATAAGTGACTAAACAAATAACTAAATAGATGGGGGTTTCTTTTAATATTATGTGTCCTAATAGTAGCATTTATTCAGATGAAAAATCAAGGGTTTTAGTGGACAAGACAAAAAGTGGAAAAGTGAGACCATGGAGAGAAAAGAAAATCGCTAATGTTGATTACTTTGAACTTCTGCATATTCTTGAATTTAAAAAGGCTGAAAGAGTAAAAGATTGTGCTGAAATATTAGAGTATAAACAAAATCGTGAAACAGGCGAAAGAAAGTTGTATCGAGTGTGGTTTTGTAAATCCAGGCTTTGTCCAATGTGCAACTGGAGGAGAGCAATGAAACATGGCATTCAGTCACAAAAGGTTGTTGCTGAAGTTATTAAACAAAAGCCAACAGTTCGTTGGTTGTTTCTCACATTAACAGTTAAAAATGTTTATGATGGCGAAGAATTAAATAAGAGTTTGTCAGATATGGCTCAAGGATTTCGCCGAATGATGCAATATAAAAAAATTAATAAAAATCTTGTTGGTTTTATGCGTGCAACGGAAGTGACAATAAATAATAAAGATAATTCTTATAATCAGCACATGCATGTATTGGTATGTGTGGAACCAACTTATTTTAAGAATACAGAAAACTACGTGAATCAAAAACAATGGATTCAATTTTGGAAAAAGGCAATGAAATTAGACTATGATCCAAATGTAAAAGTTCAAATGATTCGACCGAAAAATAAATATAAATCGGATATACAATCGGCAATTGACGAAACTGCAAAATATCCTGTAAAGGATACGGATTTTATGACCGATGATGAAGAAAAGAATTTGAAACGTTTGTCTGATTTGGAGGAAGGTTTACACCGTAAAAGGTTAATCTCCTATGGTGGTTTGTTAAAAGAAATACATAAAAAATTAAACCTTGATGACACAGAAGAAGGCGATTTGATTCATACAGATGATGACGAAAAAGCCGATGAAGATGGATTTTCTATTATTGCAATGTGGAATTGGGAACGGAAAAATTATTTTATTAAAGAGTAGTTCAACAAACGGGCCAGTTTGTTGAAGATTAGATGCTATAATTGTTATTAAAAGGATTGAAGGATGCTTAGGAAGACGAGTTATTAATAGCTGAATAAGAACGGTGCTCTCCAAATATTCTTATTTAGAAAAGCAAATCTAAAATTATCTGAAAAGGGAATGAGAATAGTGAATGGACCAATAATAATGACTAGAGAAGAAAGAATGAAGATTGTTCATGAAATTAAGGAACGAATATTGGATAAATATGGGGATGATGTTAAGGCTATTGGTGTTTATGGCTCTCTTGGTCGTCAGACTGATGGGCCCTATTCGGATATTGAGATGATGTGTGTCATGTCAACAGAGGAAGCAGAGTTCAGCCATGAATGGACAACCGGTGAGTGGAAGGTGGAAGTGAATTTTGATAGCGAAGAGATTCTACTAGATTATGCATCTCAGGTGGAATCAGATTGGCCGCTTACACATGGTCAATTTTTCTCTATTTTGCCGATTTATGATTCAGGTGGATACTTAGAGAAAGTGTATCAAACTGCTAAATCGGTAGAAGCCCAAACGTTCCACGATGCGATTTGTGCCCTTATCGTAGAAGAGCTGTTTGAATATGCAGGCAAATGGCGTAATATTCGTGTGCAAGGACCGACAACATTTCTACCATCCTTGACTGTACAGGTAGCAATGGCAGGTGCCATGTTGATTGGTCTGCATCATCGCATCTGTTATACGACGAGCGCTTCGGTCTTAACTGAAGCAGTTAAGCAATCAGATCTTCCTTCAGGTTATGACCATCTGTGCCAGTTCGTAATGTCTGGTCAACTTTCCGACTCTGAGAAACTTCTGGAATCGCTAGAGAATTTCTGGAATGGGATTCAGGAGTGGACAGAACGACACGGATATATAGTGGATGTGTCAAAACGCATACCATTTTGAACGATGACCTCTAATAATTGTTAATCATGTTGGTTACGTATTTATTAACTTCTCCTAGTATTAGTAATTATCATGGCTGTCATGGCGCATTAACGGAATAAAGGGTGTGCTTAAATCGGGCCATTTTGCGTAATAAGAAAAAGGATTAATTATGAGCGAATTGAATTAATAATAAGGTAATAGATTTACATTAGAAAATGAAAGGGGATTTTATGCGTGAGAATGTTACAGTCTATCCCGGCATTGCCAGTCGGGGATATTAAAAAGAGTATAGGTTTTTATTGCGATAAACTAGGTTTCACTTTGGTTCACCATGAAGATGGATTCGCAGTTCTAATGTGTAATGAGGTTCGGATTCATCTATGGGAGGCAAGTGATGAAGGCTGGCGCTCTCGTAGTAATGATTCACCGGTTTGTACAGGTGCGGAGTCGTTTATTGCTGGTACTGCTAGTTGCCGCATTGAAGTAGAGGGAATTGATGAATTATATCAACATATTAAGCCTTTGGGCATTTTGCACCCCAATACATCATTAAAAGATCAGTGGTGGGATGAACGAGACTTTGCAGTAATTGATCCCGACAACAATTTGATTAGCTTTTTTCAACAAATAAAAAGCTATAATCTATTATTAATCTGTTCAGCAATCGGGCGCGATTGCTGAATAAAAGATACGAGAGACCTCTCTTGTATCTTTTTTATTTTGAGTGGTTTTGTCCGTTACACTAGAAAACCGAAAGACAATAAAAATTTTATTCTTGCTGAGTCTGGCTTTCGGTAAGCTAGACAAAACGGACAAAATAAAAATTGGCAAGGGTTTAAAGGTGGAGATTTTTTGAGTGATCTTCTCAAAAAATACTACCTGTCCCTTGCTGATTT

Annotation:
Pveg: 23-259 (Forward)
RBS D4E1: 479-490 (forward)
RBS Metch: 260-271 (forward)
Terminator: 1203-1282 (forward)
Ampicillin resistance (E. coli): 2265-2924 (reverse)
D4E1 : 614-673 (forward)
amyE for D4E1 : 491-613 (forward)
Cutted Metchnikowin : 401-478 (forward)
amyE for cutted Metchnikowin: 278-400 (forward)
Bleomycin resistance (B. subtilis): 5903-6243 (forward)
ColE1 origin (E. coli): 1485-2167 (reverse)
Kanamycin resistance (B. subtilis): 4910-5680 (forward)
Prefix: 1-22 (forward)
repU (Bacillus subtilis origin): 3746-4750 (forward)
Suffixe: 986-1006 (forward)
Verification forward (VF2) primer binding site : 3247-3266 (forward)
Verification reverse (VR) primer binding site : 1386-1405 (forward)