KU Leuven 2008

Implemented kill switches: 1

Kill Switch 1

Toxins

Name Number Comment
ccdB BBa_K145151 CcdB protein is a topoisomerase poison from Escherichia coli. It is responsible for killing plasmid-free segregants and interferes with the activity of DNA gyrase. It acts to inhibit partitioning of the chromosomal DNA.

Antagonists

Name Number Comment

Topology

Kill Switch topology

Parts

Name Number Comment
Constitutive promoter BBa_J23116 BBa_J23116 is a constitutive promotor and member of the BBa_J23xxx family promotors.
RBS.3 (medium) BBa_B0032 Weak1 RBS based on Ron Weiss thesis. Efficiency of 0.3.
Double terminator BBa_B0015 The mfold results are annotated with the location of the subparts BBa_B0010 and BBa_B0012 and the BioBrick assembly scar.
tetracycline repressor from transposon Tn10 (+LVA) BBa_C0040 Coding region for the TetR protein without the Ribosome Binding Site. Modified with an LVA tail for rapid degradation of the protein and faster fall time for the emission. TetR binds to the pTet regulator (Part:BBa_R0040). aTc (anhydrotetracycline) binds to TetR and inhibits its operation.
TetR repressible promoter BBa_R0040 Sequence for pTet inverting regulator. Promoter is constitutively ON and repressed by TetR. TetR repression is inhibited by the addition of tetracycline or its analog aTc.
key3c BBa_J23008 Encodes a short RNA sequence that "unlocks" the biobrick part J01122 (lock3) which is locked RFP.
key3d BBa_J23009 riboregulator for lock3 variants.
T7 RNA Polymerase with UmuD derived tag BBa_K145014 T7 RNA Polymerase will start the transcription from a T7 promoter (such as BBa_I712074). It is tagged with a UmuD tag (at the N-terminal) for faster degradation.
T7 promoter (strong promoter from T7 bacteriophage) BBa_I712074 T7 promoter is very specific promoter which is transcribed only by specific T7 RNA polymerase. Usually this promoter is used in expression systems where T7 promoter is cotransfected with T7 RNA polymerase. That ensures strong transcription of desired genes.
lock3i BBa_J23078 lock3d derivative with a 5' extension. This is the basic part and J23077 is the RFP reporter.
LuxI Required for the synthesis of OHHL (N-(3-oxohexanoyl)-L-homoserine lactone) also known as VAI or N-(beta-ketocaproyl)homoserine lactone or 3-oxo-N-(tetrahydro-2-oxo-3-furanyl)-hexanamide an autoinducer molecule which binds to LuxR and thus acts in bioluminescence regulation.
lacI repressor from E. coli (+LVA) BBa_C0012 Coding region for the LacI protein with an LVA degradation tail and without an RBS. LacI binds to the pLac regulator BBa_R0010 and PLlac01 hybrid regulator BBa_R0011 and inhibits transcription. IPTG (Isopropylthiogalactoside) binds to LacI and inhibits its operation therefore promoting transcription. A rapid degradation tail (LVA) has been added to improve the switch time for High to Low performance of this part.
Promoter (lacI regulated and lambda pL hybrid) BBa_R0011 Inverting regulatory region controlled by LacI (BBa_C0010 and BBa_C0012 and etc.) The PLlac 0-1 promoter is a hybrid regulatory region consisting of the promoter P(L) of phage lambda with the cI binding sites replaced with lacO1. The hybrid design allows for strong promotion that can nevertheless be:repressed by LacI - the Lac inhibitor (i.e. repressor) (BBa_C0012) ([LUTZ97]). Induced by IPTG in E.Coli DH5-alpha-Z1 (same paper reference) over a >600-fold range.
autoinducer inactivation enzyme from Bacillus that hydrolyzes acetyl homoserine lactone BBa_C0060 Coding region for the autoinducer inactivation enzyme A (aiiA) LVA tagged (for untagged version see Part:BBa_C0160. The gene was originally isolated from Bacillus sp. 240B1 and it encodes an enzyme that catalyzes the degradation of N-acyl-homoserine lactones (AHLs)--quorum sensing autoinducers.
luxR repressor/activator BBa_C0062 In complex with HSL the LuxR binds to the Lux promoter and activates transcription from Pr BBa_R0062 and repressing transcription from Pl BBa_R0063.
Hybrid promoter: HSL-LuxR activated and P22 C2 repressed BBa_K145150 Hybrid promoter consisting of the Lux box upstream the -35 box and binding sites for P22 C2; OR2 and OR1 located between the -35 and -10 boxes and downstream of them respectively. Transcription from this promoter can be activated by a LuxR-HSL complex and repressed by p22 C2. Background transcription from this pomoter (no activation and no repression) is very low.
RBS (Elowitz 1999) BBa_B0034 RBS based on Elowitz repressilator.
double terminator (B0012-B0011) BBa_B0014 The mfold results are annotated with the location of the subparts BBa_B0012 and BBa_B0011.
ccdB coding region BBa_K145151 Coding region for the ccdB (control of cell death) gene. This part will cause Top10 cells to die.
RBS.4 (weaker) BBa_B0033 Weaker RBS based on Ron Weiss thesis. Strengths relative to BBa_B0030 and BBa_B0031 and BBa_B0032.
cI repressor from phage 434 (+LVA) BBa_C0052 The 434 cI repressor protein coding sequence is a 710 base-pair sequence with the standard RBS-compatible BioBrick prefix and the standard BioBrick suffix sections on its ends. It binds to the 434 regulatory sequence BBa_R0052. The sequence contains a LVA tag for faster degradation and has no RBS.
Promoter (p22 cII regulated) BBa_R0053 The p22 cII regulatory region sequence is a 97 base-pair sequence with the standard BioBrick prefix and suffix sections on its ends. p22 cII repressor protein BBa_C0053 binds to it.
c2 repressor from Salmonella phage P22 (+LVA) BBa_C0053 The P22 c2 repressor protein coding sequence is a 720 base-pair sequence with the standard RBS-compatible BioBrick prefix and the standard BioBrick suffix sections on its ends. It binds to the P22 c2 regulatory sequence BBa_R0053. The sequence contains a LVA tag for faster degradation.
Standard Promoter (434 cI regulated) BBa_R1052 The 434 cI regulatory region sequence is a 89 base-pair sequence with the standard BioBrick prefix and suffix sections on its ends. 434 cI repressor protein BBa_C0052 binds to it.
cI repressor from phage 434 (no LVA) BBa_C0056
antisense LuxI BBa_K145013 This part is a RNA that is the complement of luxI (part BBa_C0061). It will bind to the mRNA of luxI and thereby repress the translation of luxI. So in the presence of this antisense luxI the enzym luxI will not be produced.