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
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.
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
The device consist of temperature promotor and hokD coding gene.
This device is a combination of two parts submitted by previous igem teams that one （BBa_K608351） is a temperature-regulated promoter followed by the other （BBa_K1497008 ）that a gene coding for a protein hokD which induces cell lysis. The promoter we chose won???t start translation until the temperature is raised to 37℃. And at 42℃ can we get the peck efficiency of expression. The promoter is in fact a composite sequence that preceding the regulated promoter is the control sequence which can synthesis λ cI repressor. λ cI repressor binds with the promoter sequence when environment temperature is 28~30℃ while at 42℃, the protein denatures and falls off by which initiate the transcription. We use the temperature-regulated promoter to express hokD gene. The hokD gene of??E. coli??transcribes for a small polypeptide that causes cell death by elimination of vital cell wall functions. In short , our device can induce cell lysis when temperature is raised to 42℃. To deliver the antimicrobial peptides in the right place and at the right time, we designed and constructed this temperature-regulated device. When put under the sole of feet, our peptide-expressing E.coli cells in the insole would be incubated at a warmer environment due to body temperature. We use this promoter to control the synthesis of hokD for the raised temperature will promote the translation of hokD which following the promoter.
We use pSB1C3 vector to express this device for its more avaliable.
we acquired that two parts BBa_K608351 and BBa_K1497008 we based on on igem distribution plates and constructed the composite device through molecular biology technique.
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]
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
{< 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
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 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
R Protein (Endolysin: Phage lambda lysozyme)
S' Protein (Holin) / S Protein (Anti-holin)
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
The Cell Lysis Cassette consists of an S, R, and Rz protein. When paired with an activator, the cassette is expressed and cell lysis is induced. The S105 strain of the cassette has the initial 6 base pairs at the beginning of the wild type sequence removed. This causes cell lysis to be induced at a faster rate than the wild type.
This part was graciously donated to the Brown iGEM Team by Ry Young at Texas A&M.
{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
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.
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
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.
PotB59, chimeric protein that is formed by the N-Terminal region of PomB and the C-terminal of E. coli MotB It was shown that a chimeric protein, PotB, renamed as PotB59, that has the N-terminal region of PomB (1???50) fused to the C-terminal periplasmic region of E. coli MotB (59???308), is functional with PomA in E. coli as well as in strains of V. alginolyticus that are defective in motX or motY (Asai et al. 2003) Instead of the hydrogen-dependant MotA MotB Stator protein partially adapted from the V. alginolyticus, sodium-dependant Stator proteins were installed. After this adaptation, the flagellar velocity increased by twice fold.
This part contains an additional BamHI restriction site at beginning of TlpB sequence and a XhoI restriction site at the end.
{< 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
This part causes cell death. It contains holin/antiholin, endolysin and Rz/Rz1 genes.
We made point mutation and prepared wild type phage DNA.
λ -Hind Ⅲ digest (product of TaKaRa Bio)
{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
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
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
{ 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