Granulysin, a T Cell Product,Kills Bacteria by Altering Membrane Permeability
Granulysin, a protein located in the acidic granules of human NK cells and cytotoxic T cells, has microbial activity against a broad spectrum of microbial pathogens. The substance released by cytotoxic T cells (CD8) when attached to infected body cells. It creates holes in the target cell membrane and destroy it. That is,Granulysin induces apoptosis in target cells and have antimicrobial action. It is broadly antimicrobial, killing microbes that cause, for example, tuberculosis and malaria, and can destroy some tumors. A series of peptides generated from the amino acid sequence of granulysin are potential antibiotics. Granulysin increased the permeability of bacterial membranes, as judged by its ability to allow access of cytosolic ??-galactosidase to its impermeant substrate.Granulysin functions to create holes in the target cell membrane and destroy it. Granulysin is able to induce apoptosis in target cells and also has antimicrobial action.
Granulysin can be purified via nickel affinity chromatography.
Granulysin is a member of the saposin-like protein (SAPLIP) family. The gene was synthesized at GENEART in pMA vector
T4 holin, without start or stop codon, berkeley standard
T4 holin gene without start or stop codons, in BBb format. ---- This part is in BBb Format. It is flanked by BamHI and BglII sites instead of XbaI and SpeI. More information about the BBb Format is available at: [http://openwetware.org/wiki/Template:AndersonLab:BBb_Standard BBb Standard Description Page]
PCR bx3/bx4 on Bjh1301 (682 bp, EcoRI/BamHI/DpnI) Sub into pBca1256 (EcoRI/BamHI) Product is pBca1256-K112003 ---------------------------------------- bx3 Forward Biobricking of {<T4 Holin>} ccagtGAATTCatgAGATCTgcagcacctagaatatcattttcgccc bx4 Reverse Biobricking of {<T4 Holin>} cgttaGGATCCtttagcccttcctaatattctggcc
Barstar; Barnase immunity protein is under Plac(BBa_R0011)
Lysozyme and holin production are driven by pBad, and antiholin is driven by PconC5.
Assembled using 2 antibiotic assembly from basic parts.
lambda lysozyme ready to have an rbs attached with a stop codon More information about the BBb Format is available at: [http://openwetware.org/wiki/Template:AndersonLab:BBb_Standard BBb Standard Description Page]
PCR al0005/al0002 on pSB3C6-Bjh1501 (512bp, EcoRI/BamHI/DpnI) Sub into pBca1256 (EcoRI/BamHI, 2472+910, L) Product is pBca1256-K112304 ----------------------------------------------- al0005 Forward Biobricking of lambda lysozyme with spacer CCATAGAATTCatgAGATCTgaagatggtagaaatcaataatcaacgtaaggcgttcctcg al0002 Reverse Biobricking of lambda lysozyme with spacer CGttaGGATCCtcatacatcaatctctctgaccgttccgcc
T4 antiholin gene with start and stop codons, in BBb format. ---- This part is in BBb Format. It is flanked by BamHI and BglII sites instead of XbaI and SpeI. More information about the BBb Format is available at: [http://openwetware.org/wiki/Template:AndersonLab:BBb_Standard BBb Standard Description Page]
PCR bx7/bx8 on Bjh1303 (325 bp, EcoRI/BamHI/DpnI) Sub into pBca1256 (EcoRI/BamHI) Product is pBca1256-K112006 ---------------------------------------- bx7 Forward biobricking of {T4 Antiholin!} ccagtGAATTCatgAGATCTatggccttaaaagcaacagcac bx8 Reverse biobricking of {T4 Antiholin!} cgttaGGATCCtcattcagtctccaatttaatgttc
Barnase and GFP is co-expressed by luxR/AHL complex.
GoldenGate compatible Bax - cell death inducer
BAX coding sequence (Bcl-2-associated X) protein, a pro-apoptotic member of the Bcl-2 protein family which induces cell death. The part has been made compatible with the GoldenGate MoClo Assembly Standard and is free from BsaI and BpiI recognition sequences. Codon-optimised for expression in dicotyledenous plants.
Domestication for GoldenGate by removal of internal BsaI and BpiI recognition sequences. Codon-optimised for expression in dicotyledenous plants.
T4 holin gene without a start codon with a stop codon, in BBb format. ---- This part is in BBb Format. It is flanked by BamHI and BglII sites instead of XbaI and SpeI. More information about the BBb Format is available at: [http://openwetware.org/wiki/Template:AndersonLab:BBb_Standard BBb Standard Description Page]
{lambda antiholin!}; adheres to Berkeley standard
lambda antiholin with start and stop codons. This part is actually in BBb format. ---- This part is in BBb Format. It is flanked by BamHI and BglII sites instead of XbaI and SpeI. More information about the BBb Format is available at: [http://openwetware.org/wiki/Template:AndersonLab:BBb_Standard BBb Standard Description Page]
PCR al0013/al0008 on pSB3C6-Bjh1503 (355bp, EcoRI/BamHI/DpnI) Sub into pBca1256 (EcoRI/BamHI, 2472+910, L) Product is pBca1256-K112312 ----------------------------------------------- al0013 Forward Biobricking of lambda antiholin with start and stop codons CCATAGAATTCatgAGATCTatgaagCtgccagaaaaacatgacc al0008 Reverse Biobricking of lambda antiholin with start and stop codons CGttaGGATCCttattgatttctaccatcttctactccggc
lambda holin with stop codon and no start codon in BBb format More information about the BBb Format is available at: [http://openwetware.org/wiki/Template:AndersonLab:BBb_Standard BBb Standard Description Page]
PCR al0009/al0008 on pSB3C6-Bjh1502 (346bp, EcoRI/BamHI/DpnI) Sub into pBca1256 (EcoRI/BamHI, 2472+910, L) Product is pBca1256-K112307 ----------------------------------------------- al0009 Forward Biobricking of lambda holin with stop codon and no start codon CCATAGAATTCatgAGATCTccagaaaaacatgacctgttggcc al0008 Reverse Biobricking of lambda holin with stop codon and no start codon CGttaGGATCCttattgatttctaccatcttctactccggc
Lysis device based on T4 holin and lysozyme, driven by Pbad. ---- This part is in BBb Format. It is flanked by BamHI and BglII sites instead of XbaI and SpeI. More information about the BBb Format is available at: [http://openwetware.org/wiki/Template:AndersonLab:BBb_Standard BBb Standard Description Page]
A SV40 nuclear localization signal was added to the phototoxic protein KillerRed.
T4 antiholin gene with a start codon, without a stop codon, in BBb format. ---- This part is in BBb Format. It is flanked by BamHI and BglII sites instead of XbaI and SpeI. More information about the BBb Format is available at: [http://openwetware.org/wiki/Template:AndersonLab:BBb_Standard BBb Standard Description Page]
PCR bx7/bx10 on Bjh1303 (322 bp, EcoRI/BamHI/DpnI) Sub into pBca1256 (EcoRI/BamHI) Product is pBca1256-K112008 ---------------------------------------- bx7 Forward biobricking of {T4 Antiholin>} ccagtGAATTCatgAGATCTatggccttaaaagcaacagcac bx10 Reverse biobricking of {T4 Antiholin>} cgttaGGATCCttcagtctccaatttaatgttc
lambda antiholin with native rbs site and stop codon. This part is actually in BBb format. ---- This part is in BBb Format. It is flanked by BamHI and BglII sites instead of XbaI and SpeI. More information about the BBb Format is available at: [http://openwetware.org/wiki/Template:AndersonLab:BBb_Standard BBb Standard Description Page]
PCR al0016/al0008 on pSB3C6-Bjh1503 (375bp, EcoRI/BamHI/DpnI) Sub into pBca1256 (EcoRI/BamHI, 2472+910, L) Product is pBca1256-K112317 ----------------------------------------------- al0016 Forward Biobricking of lambda antiholin with rbs site CCATAGAATTCatgAGATCTccccttattgggggtaagacatg al0008 Reverse Biobricking of lambda antiholin with rbs site CGttaGGATCCttattgatttctaccatcttctactccggc
inclusion body induced cell lysis system
Long Description: This part consists of theCpxR responsive promoter (Part:BBa_K135000), a RBS (Part:BBa_B0034), ddpX gene (Part: BBa_K2110004), two terminators (Part:BBa_B0010, Part:BBa_B0012). The CpxR responsive promoter can response to the inclusion body and misfold protein in the periplasm. When this system coexpress with an exogenous gene like the PETase gene, the forming inclusion body will induce the expression of downstream ddpX gene and cause the cell lysis, which provides much convenience to the purification steps followed.
Prontein E is a membrane protein that inhibits MraY, an enzyme required for murein synthesis, and promotes cell lysis in a manner analogous to cell wall synthesis inhibitors like penicillin. However, it will keep the periplasmic space of Gram-negative strain intact. In this part, protein E will be expressed when L-arabinose is available.
Enterobacteria phage T4 Lysis Device - no promoter
This is the lysis device using three genes from enterobacteria phage T4: lysozyme, holin, and antiholin. Various promoters can be installed with this device to control the lysis of bacteria. Antiholin has its own constitutive promoter to prevent the formation of holin multimers from basal expression. When the device is off, higher expression of antiholin compared to holin is required for better stability of the device.
[Pcon].[Antiholin].[terminator] was assembled first, and then assembled with [T4 holin].[terminator]; Constitutive expression of antiholin is required before assembling holin.
Pubmed Accession Number: NC_000866 Enterobacteria phage T4
S.aureus killing device. Truncated lysostaphin coding sequence with IPTG inducible promoter.
Lysostaphin is a zinc metalloenzyme that has a specific lytic action against S.aureus. Lysostaphin has activities of three enzymes namely, endo-β-N-acetyl glucosamidase, N-acteyl-muramyl-L-alanine amidase, glycylglycine endopeptidase. Glycylglycine endopeptidase lyses staphylococcal cells by hydrolyzing glycylglycine bonds in the poly-glycine bridges which form cross links between glycopeptide chains in cell wall peptidoglycan of S.aureus cells. The wild-type lysostaphin gene encodes a preproenzyme, and the conversion of prolysostaphin to mature lysostaphin occurs extracellularly and involves the removal of the hydrophilic tandem repeat portion of the proenzyme. In order to directly produce mature lysostaphin, we truncate the preprolysostaphin and prolysostaphin sequence. With the inducing of IPTG, the device can erradicate S.aureus through the production and release of lysostaphin.
In order to directly produce mature lysostaphin, we truncate the preprolysostaphin and prolysostaphin sequence.
Lysozyme and holin production are driven by pBad, and antiholin is driven by PconC6.
T4 holin gene ready to have an rbs attached with a stop codon, in BBb format. ---- This part is in BBb Format. It is flanked by BamHI and BglII sites instead of XbaI and SpeI. More information about the BBb Format is available at: [http://openwetware.org/wiki/Template:AndersonLab:BBb_Standard BBb Standard Description Page]
PCR bx5/bx2 on Bjh1301 (692 bp, EcoRI/BamHI/DpnI) Sub into pBca1256 (EcoRI/BamHI) Product is pBca1256-K112004 ---------------------------------------- bx5 Forward Biobricking of {a~T4 Holin!} ccagtGAATTCatgAGATCTgtctatggcagcacctagaatatc bx2 Reverse Biobricking of {a~T4 Holin!} cgttaGGATCCttatttagcccttcctaatattctggccgcc
lambda lysozyme with start and stop codons in Bbb format
This variation uses PconB5 to drive the production of antiholin. ---- This part is in BBb Format. It is flanked by BamHI and BglII sites instead of XbaI and SpeI. More information about the BBb Format is available at: [http://openwetware.org/wiki/Template:AndersonLab:BBb_Standard BBb Standard Description Page]
T4 lysozyme gene ready to have an rbs attached in front, with a stop codon, in BBb format. ---- This part is in BBb Format. It is flanked by BamHI and BglII sites instead of XbaI and SpeI. More information about the BBb Format is available at: [http://openwetware.org/wiki/Template:AndersonLab:BBb_Standard BBb Standard Description Page]
PCR bx17/bx16 on Bjh1302 (530 bp, EcoRI/BamHI/DpnI) Sub into pBca1256 (EcoRI/BamHI) Product is pBca1256-K112016 ---------------------------------------- bx17 Forward biobricking of {a~T4 Endolysin!} ccagtGAATTCatgAGATCTtattatgaatatatttgaaatgttacgtatagatg bx16 Reverse biobricking of {a~T4 Endolysin!} cgttaGGATCCtagatttttatacgcgtcccaagtgcc
{a~lambda holin!}; adheres to Berkeley standard
lambda holin ready for rbs addition and has stop codon in BBb format. More information about the BBb Format is available at: [http://openwetware.org/wiki/Template:AndersonLab:BBb_Standard BBb Standard Description Page]
PCR al0011/al0008 on pSB3C6-Bjh1502 (353bp, EcoRI/BamHI/DpnI) Sub into pBca1256 (EcoRI/BamHI, 2472+910, L) Product is pBca1256-K112310 ----------------------------------------------- al0011 Forward Biobricking of lambda holin with spacer CCATAGAATTCatgAGATCTgaagatgccagaaaaacatgacctg al0008 Reverse Biobricking of lambda holin with spacer CGttaGGATCCttattgatttctaccatcttctactccggc
Holin is an essential protein for host lysis by bacteriophage. Its activity can by blocked by another protein called antiholin (BBa_K1054003). We can use this feature to prevent horizontal gene transfer of certain plasmid.
T4 antiholin gene without start and stop codons, in BBb format. ---- This part is in BBb Format. It is flanked by BamHI and BglII sites instead of XbaI and SpeI. More information about the BBb Format is available at: [http://openwetware.org/wiki/Template:AndersonLab:BBb_Standard BBb Standard Description Page]
PCR bx9/bx10 on Bjh1303 (319 bp, EcoRI/BamHI/DpnI) Sub into pBca1256 (EcoRI/BamHI) Product is pBca1256-K112009 ---------------------------------------- bx9 Forward biobricking of {<T4 Antiholin>} ccagtGAATTCatgAGATCTgccttaaaagcaacagcac bx10 Reverse biobricking of {<T4 Antiholin>} cgttaGGATCCttcagtctccaatttaatgttc
Lysis protein S from Enterobacteria phage lambda (codon usage optimized for R. opacus)
This gene codifies for a protein that makes holes in the inner membrane of the bacterial cell. A Gram positive RBS (SpoVG, http://partsregistry.org/wiki/index.php?title=Part:BBa_K143021) was added at the beginning.
Between the RBS and the CDS a spacer consisting on a scar from a standard suffix and a preffix for CDS was inserted.
The amino sequence was obtained from Uniprot (http://www.uniprot.org/uniprot/P03705#section_seq lysis protein S) and optimized for R. opacus (avoiding standard restriction sites) using the free software GeneDesigner from DNA 2.0.
Colicin-E3, its immunity protein, and colicin lysis protein is constitutively expressed strong.(under BBa_?J23101) When this device works, Colicin-E3 will be released to media, and E. coli strains which does not have immunity protein will be killed.
T4 lysozyme gene without a start codon, with a stop codon, in BBb format. ---- This part is in BBb Format. It is flanked by BamHI and BglII sites instead of XbaI and SpeI. More information about the BBb Format is available at: [http://openwetware.org/wiki/Template:AndersonLab:BBb_Standard BBb Standard Description Page]
PCR bx15/bx14 on Bjh1302 (523 bp, EcoRI/BamHI/DpnI) Sub into pBca1256 (EcoRI/BamHI) Product is pBca1256-K112013 ---------------------------------------- bx15 Forward biobricking of {<T4 Endolysin!} ccagtGAATTCatgAGATCTaatatatttgaaatgttacgtatagatg bx14 Reverse biobricking of {<T4 Endolysin!} cgttaGGATCCttatagatttttatacgcgtcccaagtgcc
The plasmid has a suicide gene and a mutation-controller sequence. It can hence eliminate devious mutations and help to restrain biobricks to only our engineered organisms.
Do not use commonly used commercial competent cells when using this plasmid. Special strains are required.
Antiholin binds holin and prevents holin multimers from forming pores on the inner membrane of bacteria.
Higher constitutive expression of antiholin compared to the expression of holin is required for the stable lysis device.
Enterobacteria phage T4 http://www.ncbi.nlm.nih.gov/entrez/viewer.fcgi?val=29366675&from=59202&to=59495&view=gbwithparts
The ccd operon having a constitutive promoter and the ccdB coding region. ccdB protein is lethal in normal cloning strains. This part is used to aid the process of moving a BioBrick part to a new plasmid. A target plasmid carrying P1010 is cut and mixed with the cut insert. Plasmids that recombine are selected against because they kill the cell that picks them up. P1010 is only provided as plasmid or in DB3.1 which is ccdB resistant.
Mouse guanylate kinase - tymidine kinase SR39 (Fusion protein mGMK_SR39)
Barnase and GFP is co-expressed by IPTG induction.
{< lambda antiholin!}; adheres to Berkeley standard
lambda antiholin with stop codon and no start codon. This part is actually in BBb format. ---- This part is in BBb Format. It is flanked by BamHI and BglII sites instead of XbaI and SpeI. More information about the BBb Format is available at: [http://openwetware.org/wiki/Template:AndersonLab:BBb_Standard BBb Standard Description Page]
PCR al0014/al0008 on pSB3C6-Bjh1503 (352bp, EcoRI/BamHI/DpnI) Sub into pBca1256 (EcoRI/BamHI, 2472+910, L) Product is pBca1256-K112313 ----------------------------------------------- al0014 Forward Biobricking of lambda antiholin with stop codon and no start codon CCATAGAATTCatgAGATCTaagCtgccagaaaaacatgacctg al0008 Reverse Biobricking of lambda antiholin with stop codon and no start codon CGttaGGATCCttattgatttctaccatcttctactccggc
{a~lambda antiholin!}; adheres to Berkeley standard
lambda antiholin ready for rbs addition and has stop codon. This part is actually in BBb format. ---- This part is in BBb Format. It is flanked by BamHI and BglII sites instead of XbaI and SpeI. More information about the BBb Format is available at: [http://openwetware.org/wiki/Template:AndersonLab:BBb_Standard BBb Standard Description Page]
PCR al0015/al0008 on pSB3C6-Bjh1503 (359bp, EcoRI/BamHI/DpnI) Sub into pBca1256 (EcoRI/BamHI, 2472+910, L) Product is pBca1256-K112316 ----------------------------------------------- al0015 Forward Biobricking of lambda antiholin with spacer CCATAGAATTCatgAGATCTagacatgaagCtgccagaaaaacatgacc al0008 Reverse Biobricking of lambda antiholin with spacer CGttaGGATCCttattgatttctaccatcttctactccggc
This variation uses PconG6 to drive the production of antiholin. ---- This part is in BBb Format. It is flanked by BamHI and BglII sites instead of XbaI and SpeI. More information about the BBb Format is available at: [http://openwetware.org/wiki/Template:AndersonLab:BBb_Standard BBb Standard Description Page]
T4 antiholin gene with a start codon, with a stop codon, in BBb format. ---- This part is in BBb Format. It is flanked by BamHI and BglII sites instead of XbaI and SpeI. More information about the BBb Format is available at: [http://openwetware.org/wiki/Template:AndersonLab:BBb_Standard BBb Standard Description Page]
PCR bx9/bx8 on Bjh1303 (322 bp, EcoRI/BamHI/DpnI) Sub into pBca1256 (EcoRI/BamHI) Product is pBca1256-K112007 ---------------------------------------- bx9 Forward biobricking of {<T4 Antiholin!} ccagtGAATTCatgAGATCTgccttaaaagcaacagcac bx8 Reverse biobricking of {<T4 Antiholin!} cgttaGGATCCtcattcagtctccaatttaatgttc
J23103 promoer - RBS B0034-RFP E1010 - Holin K112805 - endolysin K112806
A composite part comprising of a J23100 promotor and PA2652 translational unit encoding extracellular chemoreceptor
{ lambda antiholin >}; adheres to Berkeley standard
lambda antiholin with start codon and no stop codon. This part is actually in BBb format. ---- This part is in BBb Format. It is flanked by BamHI and BglII sites instead of XbaI and SpeI. More information about the BBb Format is available at: [http://openwetware.org/wiki/Template:AndersonLab:BBb_Standard BBb Standard Description Page]
PCR al0013/al0010 on pSB3C6-Bjh1503 (352bp, EcoRI/BamHI/DpnI) Sub into pBca1256 (EcoRI/BamHI, 2472+910, L) Product is pBca1256-K112314 ----------------------------------------------- al0013 Forward Biobricking of lambda antiholin with start codon and no stop codon CCATAGAATTCatgAGATCTatgaagCtgccagaaaaacatgacc al0010 Reverse Biobricking of lambda antiholin with start codon and no stop codon CGttaGGATCCttgatttctaccatcttctactccggc
lambda holin with start and stop codons in BBb format. More information about the BBb Format is available at: [http://openwetware.org/wiki/Template:AndersonLab:BBb_Standard BBb Standard Description Page]
PCR al0007/al0008 on pSB3C6-Bjh1502 (349bp, EcoRI/BamHI/DpnI) Sub into pBca1256 (EcoRI/BamHI, 2472+910, L) Product is pBca1256-K112306 ----------------------------------------------- al0007 Forward Biobricking of lambda holin with start and stop codons CCATAGAATTCatgAGATCTatgccagaaaaacatgacctgttgg al0008 Reverse Biobricking of lambda holin with start and stop codons CGttaGGATCCttattgatttctaccatcttctactccggc
T4 holin gene with a native rbs and a stop codon, in BBb format. ---- This part is in BBb Format. It is flanked by BamHI and BglII sites instead of XbaI and SpeI. More information about the BBb Format is available at: [http://openwetware.org/wiki/Template:AndersonLab:BBb_Standard BBb Standard Description Page]
PCR bx6/bx2 on Bjh1301 (704 bp, EcoRI/BamHI/DpnI) Sub into pBca1256 (EcoRI/BamHI) Product is pBca1256-K112005 ---------------------------------------- bx6 Forward Biobricking of {rbs.T4 Holin!} ccagtGAATTCatgAGATCTcttaaaaggagggtctatggcagcacctagaatatc bx2 Reverse Biobricking of {rbs.T4 Holin!} cgttaGGATCCttatttagcccttcctaatattctggccgcc
Endolysin is a lysozyme which cleaves the glycosidic bond between the C-1 of N-acetylmuramic acid (NAM) and the C-4 of N-acetylglucosamine (NAG) in the bacterial peptidoglycan (PG). This leads to the complete lysis of the cell. Holin is a protein that assembles together to form pores on inner membrane of bacteria allowing lysozyme to reach periplasm and degrade peptidoglycan layer. It is sourced from T4 bacteriophage. This composite part can be used to induce self-destruction with a regulatory device in the upstream region.
There is no RBS in the upstream region, so during the assemble procedure, this must be taken care of.
Gene sequences of endolysin and holin are gathered from the Registry.
Colicin-E3 cleave 16SrRNA at a specific site. This activity is inhibited by immunity protein for Colicin-E3(BBa_). When lysis protein for Colicin-E3(BBa_) is co-expressed, Colicin-E3 is emited to environment and kill other strain. Colicin is a type of bacteriocin produced by Escherichia coli.
lambda holin with rbs site and stop codon in BBb format. More information about the BBb Format is available at: [http://openwetware.org/wiki/Template:AndersonLab:BBb_Standard BBb Standard Description Page]
PCR al0012/al0008 on pSB3C6-Bjh1502 (369bp, EcoRI/BamHI/DpnI) Sub into pBca1256 (EcoRI/BamHI, 2472+910, L) Product is pBca1256-K112311 ----------------------------------------------- al0012 Forward Biobricking of lambda holin with rbs site CCATAGAATTCatgAGATCTattgggggtaagacCtgaag al0008 Reverse Biobricking of lambda holin with rbs site CGttaGGATCCttattgatttctaccatcttctactccggc
Lambda Lysozyme with stop codon and no start codon in BBb format
PCR al0003/al0002 on pSB3C6-Bjh1501 (505bp, EcoRI/BamHI/DpnI) Sub into pBca1256 (EcoRI/BamHI, 2472+910, L) Product is pBca1256-K112301 ----------------------------------------------- al0003 Forward Biobricking of Lambda Lysozyme with stop codon and no start codon CCATAGAATTCatgAGATCTgtagaaatcaataatcaacgtaaggcgttcctcg al0002 Reverse Biobricking of Lambda Lysozyme with stop codon and no start codon CGttaGGATCCtcatacatcaatctctctgaccgttccgcc
Lambda Lysis Cassette genes required for cell lysis. Contains holin/anti-holin, endolysin, and rz / rz1 genes.
T4 antiholin gene with a native rbs, with a stop codon, in BBb format. ---- This part is in BBb Format. It is flanked by BamHI and BglII sites instead of XbaI and SpeI. More information about the BBb Format is available at: [http://openwetware.org/wiki/Template:AndersonLab:BBb_Standard BBb Standard Description Page]
PCR bx12/bx8 on Bjh1303 (339 bp, EcoRI/BamHI/DpnI) Sub into pBca1256 (EcoRI/BamHI) Product is pBca1256-K112011 ---------------------------------------- bx12 Forward biobricking of {rbs.T4 Antiholin!} ccagtGAATTCatgAGATCTgataggaggcctttatggccttaaaagcaacagc bx8 Reverse biobricking of {rbs.T4 Antiholin!} cgttaGGATCCtcattcagtctccaatttaatgttc
Lysozyme and holin production are driven by pBad, and antiholin is driven by PconB5.
Made using 2 antibiotic assembly from basic parts.
xylR+PxylA+RBS+ydcE+Ptms+RBS+ydcD: Cell Growth Inhibition Device
CcdB is a toxin that poisons and kills E. coli by inhibiting the DNA gyrase and inducing double strand breaks. CcdB activity can be inhibited by the CcdA antitoxin. This gene is under the control of a galactose inducible promoter
CcdB is toxic so it has to be expressed in conjunction with CcdA or not on a galactose-containing medium.
T4 holin, without stop codon, berkeley standard
T4 holin gene with a start codon, without a stop codon, in BBb format. ---- This part is in BBb Format. It is flanked by BamHI and BglII sites instead of XbaI and SpeI. More information about the BBb Format is available at: [http://openwetware.org/wiki/Template:AndersonLab:BBb_Standard BBb Standard Description Page]
Lactose inducible lysis cassette expression
Constitutive LacI expression ensures tight repression of lysis.
Lysis cassette is cloned from Lambda Zap II genomic DNA.
KillerRed is a red fluorescent protein that produces reactive oxygen species (ROS) in the presence of yellow-orange light (540-585 nm). KillerRed is engineered from Aequorea victoria GFP (avGFP) to be phototoxic. It has been shown that KillerRed produces superoxide radical anions by reacting with water. Superoxide reacts with the chromophore of KillerRed, causing it to become dark, which ultimately gives rise to a bleaching effect. KillerRed is spectrally similar to mRFP1 with a similar brightness. KillerRed is oligomeric and may form large aggregates. This sequence is codon optimized for mammalian cells and has 14 rare proline codons for E. coli (CCC) and one rare arginine codon (AGA). Codon optimization should be taken into consideration if large amounts of the protein are required. Expression on a high-copy plasmid has produced detectable fluorescent signal, however.
This sequence is not codon optimized for E. coli.
Engineered from avGFP. Provided by the Bruchez lab at Carnegie Mellon
lambda lysozyme with native rbs site in BBb format. More information about the BBb Format is available at: [http://openwetware.org/wiki/Template:AndersonLab:BBb_Standard BBb Standard Description Page]
PCR al0006/al0002 on pSB3C6-Bjh1501 (527bp, EcoRI/BamHI/DpnI) Sub into pBca1256 (EcoRI/BamHI, 2472+910, L) Product is pBca1256-K112305 ----------------------------------------------- al0006 Forward Biobricking of lambda lysozyme with rbs site CCATAGAATTCatgAGATCTaaaaaagccggagtagaagatgg al0002 Reverse Biobricking of lambda lysozyme with rbs site CGttaGGATCCtcatacatcaatctctctgaccgttccgcc
{< lambda antiholin >}; adheres to Berkeley standard
lambda antiholin with no start and stop codons. This part is actually in BBb format. ---- This part is in BBb Format. It is flanked by BamHI and BglII sites instead of XbaI and SpeI. More information about the BBb Format is available at: [http://openwetware.org/wiki/Template:AndersonLab:BBb_Standard BBb Standard Description Page]
PCR al0014/al0010 on pSB3C6-Bjh1503 (349bp, EcoRI/BamHI/DpnI) Sub into pBca1256 (EcoRI/BamHI, 2472+910, L) Product is pBca1256-K112315 ----------------------------------------------- al0014 Forward Biobricking of lambda antiholin with no start and stop codons CCATAGAATTCatgAGATCTaagCtgccagaaaaacatgacctg al0010 Reverse Biobricking of lambda antiholin with no start and stop codons CGttaGGATCCttgatttctaccatcttctactccggc
The lysozyme from enterobacteria phage T4 degrades peptidoglycan layer.
High expression of lysozyme only is toxic to bacteria.
Enterobacteria phage T4 http://www.ncbi.nlm.nih.gov/entrez/viewer.fcgi?val=29366675&from=66503&to=66997&view=gbwithparts
{< lambda holin >}; adheres to Berkley standard
lambda holin with no start and stop codon in BBb format. More information about the BBb Format is available at: [http://openwetware.org/wiki/Template:AndersonLab:BBb_Standard BBb Standard Description Page]
PCR al0009/al0010 on pSB3C6-Bjh1502 (343bp, EcoRI/BamHI/DpnI) Sub into pBca1256 (EcoRI/BamHI, 2472+910, L) Product is pBca1256-K112309 ----------------------------------------------- al0009 Forward Biobricking of lambda holin with no start and stop codon CCATAGAATTCatgAGATCTccagaaaaacatgacctgttggcc al0010 Reverse Biobricking of lambda holin with no start and stop codon CGttaGGATCCttgatttctaccatcttctactccggc
Lysis inhibitor from Enterobacteria phage lambda (codon usage optimized for R. opacus)
This protein is the one in charge to avoid the formation of pores in the inner membrane of the bacterial cell. It has been optimized for expression in R. opacus. A weak RBS for Gram positive bacteria was used (http://partsregistry.org/Part:BBa_K090506:Design)in order to obtain low concentration of the protein.
We wanted a low concentration of this protein since medium to large concentrations could affect or completely inhibit the lysis device. Basal but constitutive expression of this protein is needed to avoid the formation of pores in the design lysis device.
The amino sequence was obtained from Uniprot (http://www.uniprot.org/uniprot/P03705#section_seq lysis inhibitor isoform). The codon optimization for R. opacus (avoiding standard restriction sites EcoRI, PstI, XbaI and SpeI) was made using the free software GeneDesigner from DNA 2.0.
lambda lysozyme with no start/stop codon in BBb format More information about the BBb Format is available at: [http://openwetware.org/wiki/Template:AndersonLab:BBb_Standard BBb Standard Description Page]
PCR al0003/al0004 on pSB3C6-Bjh1501 (502bp, EcoRI/BamHI/DpnI) Sub into pBca1256 (EcoRI/BamHI, 2472+910, L) Product is pBca1256-K112303 ----------------------------------------------- al0003 Forward Biobricking of lambda lysozyme with no start/stop codon CCATAGAATTCatgAGATCTgtagaaatcaataatcaacgtaaggcgttcctcg al0004 Reverse Biobricking of lambda lysozyme with no start/stop codon CGttaGGATCCtacatcaatctctctgaccgttccgc
Tlanslational unit of Colicin Lysis Protein (for colicin-E3)
Colicin lysis protein allows colicins to be released. The mechanism how the colicin lysis protein allows colicin release has not been fully elucidated, but it is sure that this protein raise membrane permiability and cause quasilysis.[1] Colicins are a cytotoxins which are released to environment and kill other related strains.
lambda lysozyme with start codon and no stop codon in BBb format. More information about the BBb Format is available at: [http://openwetware.org/wiki/Template:AndersonLab:BBb_Standard BBb Standard Description Page]
PCR al0001/al0004 on pSB3C6-Bjh1501 (505bp, EcoRI/BamHI/DpnI) Sub into pBca1256 (EcoRI/BamHI, 2472+910, L) Product is pBca1256-K112302 ----------------------------------------------- al0001 Forward Biobricking of lambda lysozyme with start codon and no stop codon CCATAGAATTCatgAGATCTatggtagaaatcaataatcaacgtaaggcgttcctcg al0004 Reverse Biobricking of lambda lysozyme with start codon and no stop codon CGttaGGATCCtacatcaatctctctgaccgttccgc
Mouse guanylate kinase - thymidine kinase TK30 (Fusion protein mGMK_TK30)
Peptidoglycan is the major constituent of cell wall of bacterial like E.coli. The D-alanyl-D-alanine dipeptidase gene (ddpX) exists in the genome of E.coli. The counterpart of ddpX, VanX, which has the same effect as ddpX, exists in many gram-positive bacterial like Enterococcus faecalis and Streptomyces toyocaensis because they need it to hydrolyze the D-Ala-D-Ala and transfer the D-Ala-D-Ala residue of peptidoglycan to D-Ala-D-Lac residue so that the antibiotic vancomycin cannot combine the peptidoglycan to cause the destruction of cell wall. However, in gram-negative bacterial like E.coli, which own the robust outer membrane that can resist the vancomycin, the hydrolase ddpX with the same effects also exists. This is strange because the gram-negative bacterial have no necessity to own this kind of seemly dangerous hydrolase. Actually the ddpX in E.coli has another vital use when they are under starvation conditions. The ddpX can hydrolyze the D-Ala-D-Ala in their cell wall to produce the D-Ala as the carbon source to maintain their life. This mechanism is only carried out when they are under starvation conditions. If the ddpX gene is overexpressed, the cell wall will be damaged and cell lysis will occur.
T4 lysozyme gene with a start codon, without a stop codon, in BBb format. ---- This part is in BBb Format. It is flanked by BamHI and BglII sites instead of XbaI and SpeI. More information about the BBb Format is available at: [http://openwetware.org/wiki/Template:AndersonLab:BBb_Standard BBb Standard Description Page]
PCR bx13/bx16 on Bjh1302 (523 bp, EcoRI/BamHI/DpnI) Sub into pBca1256 (EcoRI/BamHI) Product is pBca1256-K112014 ---------------------------------------- bx13 Forward biobricking of {T4 Endolysin>} ccagtGAATTCatgAGATCTatgaatatatttgaaatgttacg bx16 Reverse biobricking of {T4 Endolysin>} cgttaGGATCCtagatttttatacgcgtcccaagtgcc
This variation uses PconC5 to drive the production of antiholin. ---- This part is in BBb Format. It is flanked by BamHI and BglII sites instead of XbaI and SpeI. More information about the BBb Format is available at: [http://openwetware.org/wiki/Template:AndersonLab:BBb_Standard BBb Standard Description Page]
Lysis cassette[SRRz] is regulated by lactose promoter.
Because of the lysis cassette, lactose promoter must be repressed when transform it. So, we used KRX, not Top 10. This part is toxic to E.coli.
T4 lysozyme gene with start and stop codons, in BBb format. ---- This part is in BBb Format. It is flanked by BamHI and BglII sites instead of XbaI and SpeI. More information about the BBb Format is available at: [http://openwetware.org/wiki/Template:AndersonLab:BBb_Standard BBb Standard Description Page]
PCR bx13/bx14 on Bjh1302 (526 bp, EcoRI/BamHI/DpnI) Sub into pBca1256 (EcoRI/BamHI) Product is pBca1256-K112012 ---------------------------------------- bx13 Forward biobricking of {T4 Endolysin!} ccagtGAATTCatgAGATCTatgaatatatttgaaatgttacg bx14 Reverse biobricking of {T4 Endolysin!} cgttaGGATCCttatagatttttatacgcgtcccaagtgcc
T4 lysozyme with a native rbs, with a stop codon, in BBb format. ---- This part is in BBb Format. It is flanked by BamHI and BglII sites instead of XbaI and SpeI. More information about the BBb Format is available at: [http://openwetware.org/wiki/Template:AndersonLab:BBb_Standard BBb Standard Description Page]
PCR bx18/bx16 on Bjh1302 (542 bp, EcoRI/BamHI/DpnI) Sub into pBca1256 (EcoRI/BamHI) Product is pBca1256-K112017 ---------------------------------------- bx18 Forward biobricking of {rbs.T4 Endolysin!} ccagtGAATTCatgAGATCTatacttaggaggtattatgaatatatttgaaatgttac bx16 Reverse biobricking of {rbs.T4 Endolysin!} cgttaGGATCCtagatttttatacgcgtcccaagtgcc
T4 antiholin gene ready to have an rbs attached to the front, with a stop codon, in BBb format. ---- This part is in BBb Format. It is flanked by BamHI and BglII sites instead of XbaI and SpeI. More information about the BBb Format is available at: [http://openwetware.org/wiki/Template:AndersonLab:BBb_Standard BBb Standard Description Page]
PCR bx11/bx8 on Bjh1303 (329 bp, EcoRI/BamHI/DpnI) Sub into pBca1256 (EcoRI/BamHI) Product is pBca1256-K112010 ---------------------------------------- bx11 Forward biobricking of {a~T4 Antiholin!} ccagtGAATTCatgAGATCTctttatggccttaaaagcaacagc bx8 Reverse biobricking of {a~T4 Antiholin!} cgttaGGATCCtcattcagtctccaatttaatgttc
{lambda holin >}; adheres to Berkeley standard
lambda holin with start and no stop codon in BBb format. More information about the BBb Format is available at: [http://openwetware.org/wiki/Template:AndersonLab:BBb_Standard BBb Standard Description Page]
PCR al0007/al0010 on pSB3C6-Bjh1502 (346bp, EcoRI/BamHI/DpnI) Sub into pBca1256 (EcoRI/BamHI, 2472+910, L) Product is pBca1256-K112308 ----------------------------------------------- al0007 Forward Biobricking of lambda holin with start and no stop codon CCATAGAATTCatgAGATCTatgccagaaaaacatgacctgttgg al0010 Reverse Biobricking of lambda holin wtih start and no stop codon CGttaGGATCCttgatttctaccatcttctactccggc
T4 holin, complete CDS, berkeley standard
T4 holin gene, with start and stop codons. This part is actually in BBb format.
This part is the same to BBa_K358020[Lysisbox], except for its constitutive promoter[BBa_J23100]. So, this plasmid induces lambda lysis cassette more than BBa_K358020. This part also contains SΔTMD1, anti-killergene, regulated by lacP. When transform this part, you must induce the SΔTMD1 highly, do not repress lacP, so that the cell lysis will be prevented. This is a long part and a lactose operon was deleted unexpectedly in our experiments. So, we recomend you that it is better to set the cultural temperature at 30C and store this part as a plasmid, not a glycerole stock.
We used PCR to change the constitutive promoter.
Cat-SacB cassette for scarless chromosomal knockout
Cat-SacB Consists of chloramphenicol resistance marker BBa_P1004 and the Bacillus subtilis levansucrase SacB BBa_K322921
Colicin-E3, its immunity protein, and colisin lysis protein is constitutively expressed.(under BBa_J23110) When this device works, Colicin-E3 is released to media, and kill E. coli strains which does not have immunity protein.
Bacteriophage Lysis Cassette S105, R, and Rz
The gene cassette consists of the S105 protein (Holin), R protein (Endolysin), and Rz protein. When paired with an inducible promoter, the cassette is activated by the inducer and begins to produce the proteins which lyse the cell. This cassette contains the mutant S gene, which can be found individually as BBa_K124014. The S variation allows for faster lysis.
The DNA for this part was graciously donated to the Brown iGEM Team by Ry Young at Texas A&M University. The team then BioBricked the part for submission to the registry.
Lysis gene (promotes lysis in colicin-producing bacteria strain)
This lysis gene encodes for the lysis protein in colicin-producing strains of bacteria. Once activated, it causes the host cell to lyze. It also removes the immunity protein out of colicin, and hence, activates the endonuclease activity of the colicin.
T4 lysozyme without start or stop codons, in BBb format. ---- This part is in BBb Format. It is flanked by BamHI and BglII sites instead of XbaI and SpeI. More information about the BBb Format is available at: [http://openwetware.org/wiki/Template:AndersonLab:BBb_Standard BBb Standard Description Page]
PCR bx15/bx16 on Bjh1302 (520 bp, EcoRI/BamHI/DpnI) Sub into pBca1256 (EcoRI/BamHI) Product is pBca1256-K112015 ---------------------------------------- bx15 Forward biobricking of {<T4 Endolysin>} ccagtGAATTCatgAGATCTaatatatttgaaatgttacgtatagatg bx16 Reverse biobricking of {<T4 Endolysin>} cgttaGGATCCtagatttttatacgcgtcccaagtgcc