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
A SV40 nuclear localization signal was added to the phototoxic protein KillerRed.
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
A composite part between J23100 and antiholin
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.
alanine racemase (''alr'') under the control of the P''tac'' promoter
Alanin_Racemase (alr) under the control of the ptac-Promoter
pCons-TetR-pTet-TevProtease Suicide Switch
This is a kill switch part of our engineering E. Coli. pTet is constitutively ON and repressed by TetR. TetR repression is inhibited by the addition of tetracycline or its analog, aTc. In the presence of aTc TetR repression inhibited and Tev Protease produce by Ptet promoter. Tev Protease release Pexiganan from DAMP and E. Coli kill switch works succesfully.
Alanine-racemase extracted from genomic DNA from E. coli KRX. Used for our biosafety project.
Ligation via Gibson-assembly, so the primer had to have certain extensions
Part 1 of the Biosystem TetOR alive (pRha TetR alr)
Part 1 of the Biosystem M (pRha TetR alr)
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.
A DNA cassette that undergo homologous recombination into E.coli genome that contains homologous regions at each end, Kanamicin resistance gene (KanR) and death operon containing Lambda holin and lysozime genes and controlled by a Lambda cI regulated promoter
part is synthetic. holin and lysozime originates from lambda bacteriophage. the homology regions were taken from E.coli BL21 genome
Part 2 of the Biosafety-System L with GFP (dplac GFP)
A Kill Switch with "memory" time repressed by IPTG
We plan to establish a new E.Coli strain without lacI and MazEF system gene to avoid the inference of cell itself. We will test cI background expression level to see if we need to knock out cI. As is shown in Fig.1, Fig.2, Fig.3 and Fig.4, in the ???Stable State???, before poured into the soil, the E. Coli fertilizer is cultured in the medium with IPTG. Promoter 1 is a weak constitutive promoter, so lacI is transcribed and translated at a considerate low level. Therefore, high concentration of IPTG, which binds LacI and changes LacI???s conformation, is able to inactive LacI and open the promoter 2. Then, CI can be highly expressed to repress promoter3. Finally, mazF is inhibited. After fertilizing, no more IPTG exists in the soil to bind LacI. As a result, LacI with a tag which stabilize the protein takes time to accumulate to a certain high concentration, which is the repression threshold of promoter 2, aiming to represses promoter 2 as well as the expression of cI. The time needed to finish this progress is the designed ???memory time???. During this process, E. coli takes its own responsibility to deliver Mo. And after that, it can be killed to reduce the pollution to environment. As is shown in table 1, all the biobricks in the design are from the top 10 most used parts of iGEM to guarantee the feasibility. Name Number Description CI coding part BBa-C0051 Coding cI Promoter 1 BBa-J23116 Weak constitutive promoter Promoter 2 BBa-R0010 LacI repressible promoter Promoter 3 BBa-R0051 CI repressible promoter Table 1 Biobricks used in the design In the future, we plan to model the ???memory time??? and do more wet-experiments to confirm its feasibility. As for the modeling, we will test the expression level of promoter 1, the minimal stand of LacI to repress promoter 2 and the degradation speed of LacI. On the other hand, we will test the background expression of CI after removing IPTG to check if the CI concentration is low enough to open the promoter 3. Then we will strive to make this system more effective.
Name Number Description CI coding part BBa-C0051 Coding cI Promoter 1 BBa-J23116 Weak constitutive promoter Promoter 2 BBa-R0010 LacI repressible promoter Promoter 3 BBa-R0051 CI repressible promoter Table 1 Biobricks used in the design
Part 1 of the Biosafety-System Lac of Growth (pRha lacI alr)
Part 1 of the Biosafety-System L (pRha lacI alr)
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.
kanamicin ressistance gene (promoter+CDS)
selection for cassette integration into E.coli BL21 genome