LacI inducible Fast Response Module, using cleavable XylE
Made from parts - <bbpart>BBa_k316012</bbpart> and <bbpart>BBa_k316008</bbpart>
5' his tagged GFP-TEV linker-XylE construct is pre-made in the cell under constitutive promoter. Both Pveg promoter and spoVG RBS are best suited for maximal expression in B.subtilis. While GFP is attached to the XylE monomer via the TEV cleavable linker, the catalytic activity is low. Transcription of TEV protease allows cleavage of the linker between GFP and XylE, thus XylE is free to tetramerise into a fully functional enzyme. XylE is then able to act as described in <bbpart>BBa_k316004</bbpart> Please see 'Part Design' section for design considerations and parts used.
Standard biobrick assembly [http://partsregistry.org/Assembly:Standard_assembly]
Double terminator consisting of BBa_B0012 and BBa_B0011
Promoter Pveg and RBS spoVG for B. subtilis
Constitutive promoter veg(<bbpart>BBa_K143012</bbpart>) coupled to the strong Ribosome Binding Site spoVG(<bbpart>BBa_K143021</bbpart>) from ''B. subtilis''. Pveg-spoVG can be used in the context of a '''Ribosomes per second''' (RiPS) output generator '''To get the highest level of translation from this Promoter-RBS combination it must be connected to a coding region preceded by a coding region prefix1. A standard prefix will increase the distance between the RBS and the start codon, reducing translational efficiency.'''
The sequence of Pveg was obtained from the DBTBS1 and RBS-spoVG were obtained from papers2 and the sequence synthesised by GeneArt
N-terminus his tagged-GFP-XylE fusion protein
Existing biobrick parts with modifications using PCR primer extension
Constructed to be combined with promoter and terminator. The GFP is linked to XylE monomer subunit by a GGGSGGGS linker with the aim to render the enzyme inactive, via preventing tetramerization (it???s functional form).
Standard biobrick assembly and PCR primer extension. For full methods please see our wiki http://2010.igem.org/Team:Imperial_College_London/Strategy
The part was produced by nucleotide synthesis by mwg ??? eurofins. The synthetic RBS was designed to work with <bbpart>BBa_K316000</bbpart>.
TEV protease S219P autocatalysis resistant variant <bbpart>BBa_K316012</bbpart>. This part had been reversed for the 3' strand in order to reduce any read-through that may be caused by upstream elements. Expression of TEV protease is under control of enhanced hyperspank promoter <bbpart>BBa_K316000</bbpart>.
TEV protease is naturally found in Tobacco Etch Virus genome. This part contains the sequence codon optimized for expression in B.subtilis using mwg ??? eurofins [http://www.eurofinsdna.com/home.html] proprietary software.
Derived from luxICDABEG operon terminator of Vibrio fischeri <genbank>AF170104</genbank>.
Bidirectional transcriptional terminator consisting of a 22 bp stem-loop.
In the naturally-occuring sequence there is a mismatch in the stem of the stem loop. This can be corrected via an A->G mutation (at position 40 -- sequence coordinate/not MFOLD coordinate). The above sequence does not reflect this mutation (but the MFOLD image does). This terminator's location cannot be found using some inverted repeat detectors like PALINDROME because it is too short and contains a mismatch. This one was found with the help of Tom Knight. It lies between two coding regions that point towards eachother.
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.
Promoter veg a constitutive promoter for B. subtilis
The Pveg promoter was suggested to us by Dr. Jan-Willem Veening of Newcastle University. This sequence supplied was compared to that of the DBTBS database#3 then a section containing the binding site synthesised by Geneart.
Pveg is a constitutive promoter that constitutively expresses the P43 protein in ''B.subtilis''. Pveg contains binding sites for the ''B.sutbilis'' major sigma factor#1. Pveg in ''B.subtilis'' utilises two binding sites to cause high expression of genes#2, however our Pveg is lacking the upstream site to give a medium level of gene expression. It has been noted that the sporulation master regulatoion factor spoOA interacts with Pveg though it is not known how#3. The context with which we used the promoter Pveg is as a '''Polymerase Per Second''' (PoPS) generator.
The biobrick part was designed to include a single binding site for the ''B.subtilis major sigma factor. In addition the biobrick standard was applied to the promoter Pveg sequence.
SpoVG ribosome binding site (RBS) for B. subtilis
The sequence was taken from a previous research paper [1] and was constructed by Geneart.
Description: SpoVG is an endogenous ribosome binding site from B.subtilis. The sequence of the spoVG ribosome binding site is AAAGGUGGUGA which is complementary to the sequence UUUCCUCCACU from the 3' region of the 16s rRNA from B.subtilis. Previous research showed that the predicted binding energy of the 16s rRNA to the RBS is -19kcal 1
In order to ensure that the RBS is functional the actual ribosome binding site was maintained and the distance between the RBS and the start codon maintained. In order to conform to the biobrick standard the sequence flanking the RBS had to be changed but the distance between the promoter and RBS, and start codon and RBS was maintained.
Reverse strand coding Enhanced LacI-hyperspank promoter
DNA synthesis by MWG eurofins. The sequence was codon optimized for expression in B.subtilis using mwg ??? eurofins www.eurofinsdna.com proprietary software.
This part is a modified version of hyper-spank promoter for B.subtilis <bbpart>BBa_K143015</bbpart>. Hyper-spank promoter is repressed by transcriptional repressor LacI <bbpart>BBa_K143033</bbpart> and can be induced by addition of Isopropyl β-D-1-thiogalactopyranoside (IPTG). Constitutive expression of LacI is required for repression. Promoter Design The position and sequence of LacI binding was designed using existing knowledge. The stochastic nature of transcriptional repressors usually leads to background transcription. In order to minimise background the binding sites and the distance between them have been optimised. Stronger binding The natural LacI operator has 3 binding sites, all of which have variations in the binding sequences. Perfectly symmetric binding sequence was shown to have10-fold higher binding compared to wild type sequences. The aattgtgagc gctcacaatt sequence has been shown to be optimal for LacI binding Muller 1996 Oehler 1994. Optimal distance Due to the tetrameric nature of LacI it can simulataneously bind to multiple regions in the genome. Binding at multple sites can produce much stronger repression (muller 1996) by increasing local LacI concentrations. Due to the helical nature of DNA the distance between the operator sites plays an important role in the strength of repression. Maximal repression at 70.5bp, second strongest at 92.5bp and third at 115.5bp
Designed for minimal basal transcription by altering the LacI binding site sequence and distance between them.
TEV protease S219P autocatalysis resistant variant
TEV protease is naturally found in Tobacco Etch Virus genome. This part contains the sequence codon optimized for expression in B.subtilis using mwg ??? eurofins www.eurofinsdna.com proprietary software.
TEV protease S219P autocatalysis resistant variant Introduction : This is the nuclear inclusion protease, endogenous to Tobacco Etch Virus and is used in the late lifecycle to cleave polyprotein precursors. The recognition sequence is ENLYFQG/S [http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6W9V-45PMGK3-9P&_user=217827&_coverDate=02%2F01%2F1994&_rdoc=1&_fmt=high&_orig=search&_origin=search&_sort=d&_docanchor=&view=c&_acct=C000011279&_version=1&_urlVersion=0&_userid=217827&md5=e075aad3a349720ad9484095d01a65be&searchtype=a]] between QG or QSDue to it???s stringent sequence specificity, TEV is commonly used to cleave genetically engineered proteins. Uses: TEV proteinase is used to cleave fusion proteins. It is useful due to its high degree of specificity http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6W9V-45PMGK3-9P&_user=217827&_coverDate=02%2F01%2F1994&_rdoc=1&_fmt=high&_orig=search&_origin=search&_sort=d&_docanchor=&view=c&_acct=C000011279&_version=1&_urlVersion=0&_userid=217827&md5=e075aad3a349720ad9484095d01a65be&searchtype=a and potential to be used in vivo or in vitro applications. Auto-inactivation Wild type TEV protease also cleaves itself at Met 218 and Ser 219 http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6WXR-45R86R6-5X&_user=7635175&_coverDate=06%2F20%2F1995&_rdoc=1&_fmt=high&_orig=search&_origin=search&_sort=d&_docanchor=&view=c&_acct=C000011279&_version=1&_urlVersion=0&_userid=7635175&md5=0a1521e783c0bd4126b145f3f6d766d4&searchtype=a . This leads to auto-inactivation of the TEV protease and progressive loss of activity of the protein. The rate of inactivation is proportional to the concentration of protease http://peds.oxfordjournals.org/content/14/12/993.long More stable Mutants have been produced by single amino acid substitutions S219V (AGC(serine) to GTG(valine) and S219P (AGC(serine) to CCG(proline) Tobacco etch virus protease: mechanism of autolysis and rational design of stable mutants with wild-type catalytic proficiency '''Table I.''' Kinetic parameters for wild-type and mutant TEV proteases with the peptide substrate TENLYFQSGTRR-NH2. Enzyme Km (mM) kcat (s−1) kcat/Km (mM−1 s−1) Wild-type 0.061 ?? 0.010 0.16 ?? 0.01 2.62 ?? 0.46 S219V* 0.041 ?? 0.010 0.19 ?? 0.01 4.63 ?? 1.16 S219P* 0.066 ?? 0.008 0.09 ?? 0.01 1.36 ?? 0.22 S219P* - virtually imperivious to autocatalysis S219V* - retains same activity as wild type Full article can be seen here http://peds.oxfordjournals.org/content/14/12/993.long
The part was produced by nucleotide synthesis by mwg ??? eurofin
Conversion of the iGEM parts registry to SBOL2.1