This is a fusion protein with Ag43, a protein from E coli tagged with a 6xHis tag on the N-terminal and sfGFP tagged on C-terminal of the pro-protein. Ag43 causes cell-cell adhesion in E coli, and the 6xHis and sfGFP tags are present to allow convenient study dependence of Ag43 mediated aggregation on Ag43 expression levels, which can be determined from the sfGFP fluoresence and/or quantification of 6xHis tag. The 6xHis tag is included to allow protein concentration estimation in case the sfGFP fails to fold properly.
The location for the 6xHis tag is the same as that for BBa_K2168000(Ag43 tagged with 6xHis on N-terminal under pBAD/araC promoter) and uses the same design principles. For independent folding, a flexible GGGGSGGGGS linker was added between Ag43 and sfGFP, and the linker was choosen to be not very hydrophilic or hydrophobic, since Ag43 lies very close to lipid membranes.
This part was made from Gibson assemblies from fragments of two previous parts - BBa_K1352000 and BBa_I746908, which code for Ag43 and sfGFP respectively.
pSB1AK3 is a high copy number plasmid carrying ampicillin and kanamycin resistance. The replication origin is a pUC19-derived pMB1 (copy number of 100-300 per cell).
Expression Plasmid coding for His-tag, also containing a thrombine cleavage site between coding region and Histag, so that His-tag can be cleaved off. Vecotr opening with PstI/XmaI, clone part into it with PstI/NgoMIV. Ampicillin resistance.
very strong RBS 6xHis-Tag Thromin-cleavage-site NgoMIV-compatible restriction site for in-frame insertion
pSB1AT3 is a high copy number plasmid carrying ampicillin and tetracycline resistance. The replication origin is a pUC19-derived pMB1 (copy number of 100-300 per cell).
This is a high copy plasmid for use in assembly of BioBrick® standard biological parts.
This plasmid was derived from pSB1AK3-P1010.
Verification reverse (VR) primer binding site
Verification forward (VF2) primer binding site
pTUM102 yeast shuttle vector with pTEF2 promoter
This part corresponds to pTUM100 (BBa_K801000). The constitutive pTEF1 promoter has been cloned in front of the MCS (RFC25 prefix and suffix).
All forbidden restriction sites have been removed from the plasmid backbone, making it RFC25 compatible.
Based on invitrogen's pYES2 plasmid. For mor information see the part information of pTUM100 (BBa_K801000).
pSB1A30: High copy BioBrick cloning/expression backbone carrying Amp resitance
Constantin Ahlmann-Eltze, Charlotte Bunne, Magdalena B??scher, Jan Gleixner, Max Horn, Anna Huhn, Nils Klughammer, Jakob Kreft, Elisabeth Sch??fer, Carolin Schmelas, Silvan Schmitz, Max Waldha
37℃ induced RBS + tetR + double terminator
It is a transcriptional unit including 37℃ induced RBS + tetR + double terminator. It can be added various promoter to adapt your conditions.
To regulate following genes by temperature. To maintain cells at 37?? C ↑ : the tetR protein be generated and repress Ptet's circuit. To release cells at 37?? C ↓ : the tetR protein not be generated and do not repress Ptet's circuit.
the 37℃ induced RBS cames from iGEM08_TUDelft
Constitutive promoter + 37℃ induced RBS + tetR + double terminator
we took advantage of the interaction between 37?? C induced RBS, tetR gene and pTet promoter to create the temperature regulating circuit. The following is a description of our prescribed strategy. First, we use the constitutive promoter, 37?? C induced RBS (K115002), the tetR gene and terminators. We will call this part the ???Regulator???. We speculate that when the ???Regulator??? and ???Production??? sections are combined, we can achieve a high bacteria growth rate and close to no target protein producing rate at T>37??C, and low bacteria growth rate and high target protein producing rate at T<37??C.
To regulate following gene by temperature
pSB1C3F-Vector pSB1C3 with RFP-cassette in Freiburg Standard
To allow the possibility to save some nucleotides of primer-overhangs, you can use this vector. The two AgeI-restriction sites from the original RFP-cassette have been removed. The cassette is flanked by the modified Freiburg standard (http://2012.igem.org/wiki/images/b/b9/LMU-Munich_2012_Our_Freiburg_standard_FusionPrefix.pdf) that includes an optimal ribosome binding site between XbaI and NgoMIV restriction sites.
Two AgeI-restriction sites have been removed by two subsequent quikchange site-directed mutagnesis reactions. The RFP-cassette was PCR-amplified with the overhangs: prefix: GAATTCCGCGGCCGCTTCTAGATAAGGAGGAACTACTATGGCCGGC suffix: ACCGGTTAATACTAGTAGCGGCCGCTGCAGT
pSB1C3; the RFP-part is originally from Discosoma sp.
B0015+pBAD+vioAB+B0015+pBAD+TPP Up-regulated Hammerhead Ribozyme 1.20+vioD+B0015+pBAD+vioE
B0015+pBAD+vioAB+B0015+pBAD+TPP ribozyme 1.20+vioD+B0015+pBAD+vioE
The Ribosomal Binding Site of vioD is contained in TPP ribozyme 1.20.
The hammerhead ribozyme comes from E. coli, and the TPP aptamer comes from B. subtilis.
pTUM103 yeast shuttle vector with pADH1 promoter
This part corresponds to pTUM100 (BBa_K801000). The constitutive pADH1 promoter has been cloned in front of the MCS (RFC25 prefix and suffix).
All forbidden restriction sites have been removed from the plasmid backbone, making it RFC25 compatible .
Based on invitrogen's pYES2 plasmid. For more information see the part information of pTUM100 (BBa_K801000).
TPP Down-regulated Hammerhead Ribozyme 2.5 with Native RBS+E0040+B0015
Yangyang ZHAO, Yan GONG, Zhenrun ZHANG, Qingyang XIAO
This part is TPP up-regulated hammerhead ribozyme, consisting of 131 nucleotides.
This part contains a native ribosomal binding site
This part was an engineered RNA controller composed of a TPP aptamer from B.subtilis and a hammerhead motif.
B0015+pBAD+vioABD+B0015+pBAD+TPP Down-regulated Hammerhead Ribozyme 2.5+vioE
the Ribosomal Binding Site of vioE is contained in TPP Ribozyme.
Hammerhead Ribozyme backbone comes from E.coli, and TPP aptamer comes from B.subtilis.
pSB1AC3 is a high copy number plasmid carrying ampicillin and chloramphenicol resistance. The replication origin is a pUC19-derived pMB1 (copy number of 100-300 per cell). For the version of this plasmid with a CcdB insert, see BBa_P1010.
Silent g->a mutation found during sequencing
This a GFP reporter regulated by a self-cleaving hammerhead ribozyme. The self-cleaving of the hammerhead ribozyme would lead to the formation of the intron P1 stem, thus triggering the self-splicing of the intron.
The self-cleaving of the hammerhead ribozyme would lead to the formation of the intron P1 stem, thus triggering the self-splicing of the intron.
The intron derives from the genome of Cd
This part was derived from pSB1AC3-P1010.
pTUM104 yeast shuttle vector with GAL1 promoter
This part is based on the plasmid pTUM100 (BBa_K801000). The galactose-inducible GAL1 yeast promoter has been cloned in front of the MCS (RFC25 prefix and suffix).
All forbidden restriction sites have been removed, making this plasmid RFC25-compatible.
pSB1A3-1 is a high copy number plasmid carrying ampicillin resistance. The replication origin is a pUC19-derived pMB1 (copy number of 100-300 per cell). pSB1A3 has terminators bracketing its MCS which are designed to prevent transcription from *inside* the MCS from reading out into the vector. The efficiency of these terminators is known to be < 100%. Ideally we would construct a future set of terminators for bracketing a MCS that were 100% efficient in terminating both into and out of the MCS region. A fully annotated Genbank file is attached below.
The ampicillin resistance marker needed to be eliminated.
Austin Che constructed this plamid from pSB1AT3-P1010.
Verifiction forward (VF2) primer binding site
Compatible with the Plug'n'Play assembly standard
This a GFP reporter regulated by an allosteric intron responsive to theophylline.
The ligand response is alter from c-di-GMP to theophylline by merging a theophylline hammerhead ribozyme to the intron.
pTUM100 yeast shuttle vector based on pYES2
A yeast shuttle vector containing the following structural elements: * 2?? ori for replication in ''S. cerevisiae'' * URA3-gene for selection of transformed yeast via Uracil prototrophy * pUC ori for replication in ''E. coli'' * AmpR-gene for selection of transformed ''E. coli'' via Ampicillin resistance * f1 ori * MCS containing the RFC25 pre- and suffix for easy insertion of biobricks * following the MCS: CYC1 yeast transcriptional terminator Note: To use this backbone, a ''S. cerevisiae'' strain with Uracil-auxotrophy (such as strain INVSc1) is required for positive selection of transformants. Successfully transformed yeast cells will become prototrophic for uracil and can be selected on Agar-plates deficient of uracil.
Yan GONG, Shuobin CHEN, Yunpeng LIU, Xiaowei YAN
RFP+RBS_SDA-C1+OR21 double directed promoter+SDcro CI434+eGFP
mutate the RBS seqence change the translation rate of CI434 to get the appropriate ratio of green and red.
The part is made up of the standard parts from partsregistry, and some of the sub-parts are inverted by PCR.
Crystal protein gene (cry11Aa) from Bacillus thuringiensis subsp. Israelensis.
The cry weapon system produce crystal protein, targetting the wrigglers, larvae of mosquitoes. The cry11Aa gene is cloned from Bacillus thuringiensis subsp. Israelensis. The cry protein is controlled by the tetR-repressible promoter PtetR (BBa_R0040), which in turn is regulated by a temperature control system. When E.coli is released into environment (<37C), tetR begins to degrade, resulting in the promoter PtetR (BBa_R0011) expressing downstream genes, cry11Aa (BBa_K332012) and Green Fluorescent Protein (GFP)(BBa_E0040).
Procedures (I) Culture Bti. Bacillus thuringiensis subsp. Israelensis (BCRC15860) 1. Prepare agar plate for Bti. Beef extract 3.0g Peptone 5.0g Agar 15.0g ddH2O 1 L *adjust PH to 7.0 *No antibiotic *Be aware of contamination 2. Plate Bti. on the medium and incubate the cultures at 30??C overnight. (II) Clone cry11Aa from Bti. into TA vector 1. Extract genomic DNA of Bti. by liquid nitrogen. 2. Add some ddH2O to dilute the DNA. 3. Design primers Vf2: ATTCAATAAAAGGTGGAATGAATTATGGA Tm: 53??C VR: GTGCTAACATGACTTCTACTTTAGT Tm: 52.8??C 4. Find the best PCR condition by gradient PCR. Anneling temperature: 51??C??10??C 5. PCR by B-taq plus on the best condition Template DNA（10ng/μl） 2.0 B-taq buffer 5.0 dNTP（2mM） 5.0 forward primer（10μM） 1.5 reverse primer（10μM） 1.5 B-taq plus DNA polymerase（2Kb） 1.0 ddH2O 34.0 Total 50 μl 6. Digestion to confirm the cry11Aa fragment and enzyme sites. 7. TA clone 8. DNA sequencing (III) PCR mutagenesis at two enzyme sites --- EcoR1 and Spe1 1. Design primers by primerX. EcoR1-Vf2: CGG GTA CAA TCT CAG AAC TCG GGA AAT AAT AGA A EcoR1-VR: TTC TAT TAT TTC CCG AGT TCT GAG ATT GTA CCC G Spe1-Vf2: ATA ATG AAT GGG GAG GAC TGG TTT ATA AGT TAT TAA TGG G Spe1-VR: CTT CCC CCA TTA ATA ACT TAT AAA CTA GTC CTC CCC ATT CAT 2. Digestion to confirm the fragments (IV) PCR construction of Biobrick parts 1. Design primers by Assembly standard 10. Vf2: GTTTCTTC GAATTC GCGGCCGC T TCTAG ATGGAAGATAGTTCTTTAGATACT VR: GTTTCTTC CTGCAG CGGCCGC T ACTAGT A CTACTTTAGTAACGGATT 2. PCR condition 3. Ligation to backbones(Psb1C3). (V) Transform into E.coli 1. Thaw competent cells and BBa_K332012 plasmid on ice. 2. Add 2ul plasmid to competent cell and place in ice for 5 minutes. 3. Put the transformed cells into 42℃ water bath for 45 seconds. 4. Plate the cells on the appropriate media and antibiotic, such as agar plates with 25 ??g/ml kanamycin. 5. Incubate the cultures at 37??C overnight. (VI) Culture with mosquito larvae and observe
Crystal protein gene (cry11Aa) from Bacillus thuringiensis subsp. Israelensis. BCRC Number: 15860 Organism: Bacillus thuringiensis subsp. israelensis de Barjac History: << ATCC << USDA << ONR 60 << H. de Barjac << L. J. Goldberg 60 Other Collection No.: ATCC 35646 Source: sewage, Israel Characterization: Production of delta-endotoxin against dipteran larvae --B3009 Others: referred to as the type strain --B3008 Growth Conditions: 30??C , Medium ID: 3 Oxygen Requirement: Aerobic Biosafety Level: 1 References: Temeyer, K. B. 1984. Larvicidal activity of Bacillus thuringiensis subsp. israelensis in the dipteran Haematobia irritans. Appl. Environ. Microbiol.47 : 952-955. PMID: 6742837. Demezas, D. H. and J. Bell. 1995. Evaluation of low molecular weight RNA profiles and ribotyping to differentiate some Bacillus species. Syst. Appl. Microbiol.18 : 582-589.
This is the part we actually created to test the glue production by E. coli. HfsG and HfsH come from Caulobacter crescentu. Caulobacter crescentus is an aquatic, Gram-negative bacterium that divides asymmetrically and is able to synthetize a strong glue. This glue is mainly made of a polysaccharide. There are different proteins needed to synthetize, export and attach it to the stalk of Caulobacter. To see the hole system, please see our wiki page [1]. In our project, we would like this glue to be produced by Escherichia coli. As E. coli does have homolog genes for many of these proteins, but not for HfsG and HfsH, we decided to create a plasmid including only the genes coding for these two proteins. HfsG is a glycosyltransferase and HfsH is a carbohydrate esterase. RFP has been added as a reporter.
As many mutations were needed to make the part compatible with the standard 10, we decided to make hfsG and hfsH synthetized by GeneArt [2]. We also optimized it for E. coli. We mean that the codons were changed to favour the most present in E. coli.
HfsG and HfsH sequences come from Caulobacter crescentus.
The RBS of CI434 is CGAGACC in this bistble switch, which makes the RFP expresses three times as the GFP does in this bistable switch.
The library is constructed by mutagenesis.
pTUM101 yeast shuttle vector with pTEF1 promoter
All forbidden restriction sites have been removed from the plasmid backbone, making it RFC25 compatible
Transcriptionally insulated high copy BioBrick plasmid
pSB1A4 was constructed by adding double terminators ([BBa_B0015]) before the BioBrick prefix and after the BioBrick suffix in pSB1A3. This insulator blocks read-through transcription into BioBrick parts/ devices.
The double terminators were assembled from small smaller overlapping ssDNA oligos. The outer-most EcoRI and PstI sites were mutated while the inner-most BioBrick cut sites were restored.
Based upon pSB1A3; Includes two copies of [BBa_B0015] assembled from DNA oligos
Constitutive promoter+37℃ induced RBS+tetR+double terminator+Ptet+RBS+GFP+double terminator
We want to utilize a temperature-regulated mechanism to control the expression period of a crystal protein. Therefore, we took advantage of the interaction between 37?? C induced RBS, tetR gene and pTet promoter to create the temperature regulating circuit. The following is a description of our prescribed strategy. First, we use the constitutive promoter, 37?? C induced RBS (K115002), the tetR gene and terminators. We will call this part the ???Regulator???.
Plasmid for Plug'n'Play. Contains an Ampicillin resistance marker.
Compatible with the Plug'n'Play assembly standard.
This part encodes E coli protein Ag43, with an additional 6xHis tag for easy purification and quantification. Ag43 expression is known to cause E coli cells to adhere together, and the 6xHis tag was added to allow for convenient study of variation of E coli aggregation with respect to Ag43 expression levels.
Ag43 is an outer membrane E coli protein, so it has a N-terminal 52 amino acid long signal peptide sequence, which is cleaved after Ag43 pro-protein is transported into the periplasmic space. Since strong dependence has been demonstrated on the signal peptide cleavage on the signal peptide-protein junction, the 6xHis tag should be added after Ala53, the residue immediately after the signal peptide sequence.
This comes from Gibson assembly of fragments from a previous biobrick, BBa_K1352000 by Team:Aberdeen_Scotland, 2014.