Bxb1 integrase and Recombination Directionality Factor (RDF) Under ???Lock and Key??? Control with L
BBa_I714036, BBa_R0011, BBa_J23119 and BBa_Q04121 were obtained from the iGEM 2013 distribution kit. The rest of the biobricks were synthesized by BioBasic Inc.
Bxb1 integrase (BBa_K1039003) and lacI repressor (BBa_Q04121) are under the constitutive strong promoter BBa_J23119. Key (BBa_I714036) and Bxb1 RDF (BBa_K1039006) are under the lac inducible promoter BBa_R0011. Bxb1 integrase (BBa_K1039003) and recombination directionality factor (RDF) (BBa_K1039006) with ribosome binding sites associated with the cis-repressive RNA ???lock??? (BBa_J01010), so translation is inhibited. The trans-activating RNA ???key??? (BBa_I714036) can displace the lock and allows translation. Together, Bxb1 integrase and RDF (BBa_K1039003 and BBa_K1039006) catalyze site-specific recombination between Bxb1 attR and attL sites (Bba_K1039033 and Bba_K1039034) They will also allow flipping of Bxb1 invertible promoter switches BBa_K1039004 and BBa_K1039004 from RL state to PB state. Lock and key regulate BXB1 integrase and BRDF expression. BXB1 integrase and BRDF???s expression is blocked by lock. Key expression is controlled by a lacI repressor, upon IPTG addition to the media, key will be expressed, which then unlocks BXB1 integrase and BRDF.
Bxb1 Integrase (BBa_K1039003) has the following three silent mutations, to remove forbidden restriction sites, base 273 G->A, base 1269 A->T and base 1440 G->A
Double terminator consisting of BBa_B0010 and BBa_B0012
Biobricked version of Isaacs' riboregulator cis repressed lock, crR12.
Promoter (lacI regulated, lambda pL hybrid)
represillator of Elowitz and Leibler (2000)
Inverting regulatory region controlled by LacI (<bb_part>BBa_C0010</bb_part>, <bb_part>BBa_C0011</bb_part>, 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 tightly repressed by LacI, the Lac inhibitor (i.e. repressor) (<bb_part>BBa_C0010</bb_part>) ([LUTZ97]). The activity of the promoter can be regulated over a >600-fold range by IPTG in E.Coli DH5-alpha-Z1 (same paper reference).
hybrid promoter design to create strong promoter that is, at the same time, highly repressible. note that the upstream operator installed in this hybrid is slightly different than the one in the original source (Lutz and Bujard, 1997). the most upstream operator region is slightly truncated in the represillator version, so that both operators in the hybrid are the same sequence. see references for details. also, the sequence has been truncated after the transcriptional start site. LacI binds to this regulator. This part is incompatible with species containing active LacI coding regions. Lactose and IPTG disable the operation of LacI and increase transcription. This part is incompatible with environments containing lactose or lactose analogs.
Neelaksh Varshney, Grace Kenney, Daniel Shen, Samantha Sutton
The Isaacs key1 riboregulator taR12 is modeled on the hok/sok post-segregational killing method of plasmid R1. It makes 25 base pairs with its counterpart, the riboregulator J01010 (aka "lock 1" or "crR12"). Isaacs documentation (see reference #1): Upon induction of the araC promoter (using 0.25% arabinose solution), the taR12 key activated the corresponding crR12 lock to express the downstream indicator, GFP, to levels of fluorescence 19-fold greater than without taR12 expression. Maximal expression was reached at 70 minutes after addition of arabinose solution, and was maintained after that point.
Overlap extension of synthetic oligonucleotides
LacI QPI with strong RBS, hybrid promoter
Ghosh P, Kim AI, Hatfull GF. The orientation of mycobacteriophage Bxb1 inte- gration is solely dependent on the central dinucleotide of attP and attB. Molecular Cell 2003, 12:1101???1111.
In nature, serine integrases allow phages to integrate into the bacterial genome by site-specific recombination between attachment sites on the phage and bacterial genomes (attP and attB), both of which are between 30 and 60 bp depending which phage serine integrase is considered. Recombination between attP and attB leaves behind left and right attachment sites (attL and attR), which cannot be acted upon by Int alone. Thus, in the presence of Int alone, this recombination is irreversible (1).
Bxb1 Recombination Directionality Factor
The sequence was designed by Bonnet lab at the Department of Bioengineering, Stanford University, and was further modified by Waterloo team.
This is the recombination directionality factor (RDF) that acts with Bxb1 serine integrase. It reverses the recombination directionality of Bxb1 integrase (BBa_K1039003). In concert with Bxb1 RDF, Bxb1 integrase (BBa_K1039003) catalyzes site-specific recombination between Bxb1 attR and attL sites, to produce Bxb1 attP and attB sites. This recombination is uni-directional, i.e. attP and attB cannot be recombined to produce attL and attR in the presence of Bxb1 integrase and RDF. In concert with Bxb1 RDF (BBa_K1039006), Bxb1 integrase can be used to flip the Bxb1 invertible promoter switches BBa_K1039004 and BBa_K1039005 from RL state to PB state.
Derived from the TE terminator of T7 bacteriophage between Genes 1.3 and 1.4 <genbank>V01146</genbank>.
Transcription terminator for the E.coli RNA polymerase.
Suggested by Sri Kosuri and Drew Endy as a high efficiency terminator. The 5' end cutoff was placed immediately after the TAA stop codon and the 3' end cutoff was placed just prior to the RBS of Gene 1.4 (before AAGGAG). Use anywhere transcription should be stopped when the gene of interest is upstream of this terminator.
Transcriptional terminator consisting of a 64 bp stem-loop.
RBS (Elowitz 1999) -- defines RBS efficiency
Varies from -6 to +1 region from original sequence to accomodate BioBricks suffix. No secondary structures are formed in the given RBS region. Users should check for secondary structures induced in the RBS by upstream and downstream elements in the +50 to -50 region, as such structures will greatly affect the strength of the RBS. Contact info for this part: Brian Chow
Vinay S Mahajan, Voichita D. Marinescu, Brian Chow, Alexander D Wissner-Gross and Peter Carr IAP, 2003.
Coding region for the LacI protein with an LVA degradation tail and without an RBS. LacI binds to the pLac regulator <bb_part>BBa_R0010</bb_part> and PLlac01 hybrid regulator <bb_part>BBa_R0011</bb_part> and inhibits transcription. IPTG (Isopropylthiogalactoside) binds to LacI and inhibits its operation, therefore promoting transcription. A rapid degredation tail (LVA) has been added to improve the High to Low performance of this part.
References (unparsed) here: Elowitz, M.B., Leibler, S. A synthetic oscillatory network of transcriptional regulators. Nature 403, 335-338 (2000). [ELOW00] References (unparsed) here: Elowitz, M.B., Leibler, S. A synthetic oscillatory network of transcriptional regulators. Nature 403, 335-338 (2000). [ELOW00] Sequence taken from the repressilator of Elowitz and Leibler (2000). The obtained sequence was compared to the wild-type sequence for LacI obtained through a database search. The sequence had been modified from the wild-type in that wild-type GTG start was changed to an ATG start (note, actual ORF in E.coli has several GTG starts it would seem). The LVA tag has been added for quicker degradation. Incompatible with systems containing LacI, lactose, or IPTG.
Grace Kenney, Daniel Shen, Neelaksh Varshney, Samantha Sutton
Conversion of the iGEM parts registry to SBOL2.1