Team:Bioriidl Somaiya/Parts

Project

BioBricks Parts

The biology circuit assembly was the initial stage for the system. As without this part were would not be able to target contaminants and make them weak enough, so that our system could quickly kill the contaminants.

Basic Parts

Part:BBa_K3000020

CAP Binding site

Part:BBa_K3000021

Lacpromoter

Part:BBa_K3000023

Lac operator

Part:BBa_K3000024

6xhis tag

Part:BBa_B0034

RBS (Elowitz 1999): defines RBS efficiency

Part:BBa_K3000024

6xhis tag

Part:BBa_K3000025

T7 Terminator

Part:BBa_K3000026

ompT (Excretion Tags)

Coding Sequence

Part:BBa_K3000030

nisin-A

PPart:BBa_K3000027

Sbo-A with alb-A

Part:BBa_K3000028

Spore Killing factor(SKF) protein expressing gene

Composite Parts

BBa_K3000001

Nisin coding gene fragment

BBa_K3000002

Nisin expressing gene fragment with ompT excretion tag

BBa_K3000004

Antilisterial bacteriocin subtilosin biosynthesis protein

BBa_K3000007

SKF protein coding gene fragment

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Part:BBa_K3000020

The CAP binding site is a positive regulatory site that is bound by catabolite activator protein (CAP). When CAP is bound to this site, it promotes transcription by helping RNA polymerase bind to the promoter. So it acts as a kill switch in our system.

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Part:BBa_K3000021

This lac promoter includes a CAP binding site and the lac operator sequence lacO. The lac promoter is not as strong as the tac or the trc promoter, but in high copy-number vectors it allows expression of foreign proteins at respectable levels. In the absence of the lac repressor (LacI) expression from the lac promoter is turned on. Maximum induction of the lac promoter requires the action of the cAMP activator protein, which is most active when cells are grown in medium lacking glucose. Media that contain glucose as carbon source should not be used to express genes from this promoter. For inducible gene expression from the lac promoter the lac repressor (lacI) is necessary. The repressor binds to the operator DNA with high specificity and inhibits gene expression until an inducer like allolactose or the analog IPTG (Isopropyl-?-D-thio-galactoside) is added to the medium. The inducer binds to the repressor, causing a change in its shape. Thus altered, the repressor is unable to bind to the operator, allowing RNA polymerase (RNAP) to transcribe the following genes and thereby leading to high levels of the encoded proteins.

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Part:BBa_K3000023

The lac operon in the bacterium Escherichia coli functions by a repression mechanism in which an inhibitor protein (lacI) binds to regulatory sites (lacO) in the promoter and turns off transcription. On the addition of lactose, the lacI protein undergoes a conformational change, which changes its binding affinity for the lacO sequences. The lacI protein thereby comes off the lacO sites, and transcription can occur. E. coli uses this system to tightly control the genes required for the use of lactose, and it is completely reversible.

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Part:BBa_K3000024

The His-tag (also called 6xHis-tag) is one of the simplest and most widely used purification tags, with six or more consecutive histidine residues. These residues readily coordinate with transition metal ions such as Ni2+ or Co2+ immobilized on beads or resin for purification. So no need for additional steps during purification of protein.

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Part:BBa_B0034

A ribosome binding site, or ribosomal binding site (RBS), is a sequence of nucleotides upstream of the start codon of an mRNA transcript that will be responsible for the recruitment of a ribosome during the initiation of protein translation.

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Part:BBa_K3000024

The His-tag (also called 6xHis-tag) is one of the simplest and most widely used purification tags, with six or more consecutive histidine residues. These residues readily coordinate with transition metal ions such as Ni2+ or Co2+ immobilized on beads or resin for purification. So no need for additional steps during purification of protein.

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Part:BBa_K3000025

Phage t7 transcription terminators comprise at a minimum, the following two distinct features: a dG + dC-rich dyad-symmetrical sequence, which encodes an RNA hairpin structure, and a hairpin-distal dA + dT-rich region that encodes a continuous tract of ribouridine residues in the RNA transcript within which RNA polymerase terminates.

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Part:BBa_K3000026

OmpT is a 33.5 kDa outer membrane protein consisting of 10 antiparallel strands that are connected by 5 extracellular loops. The antiparallel strands form a beta-barrel structure that spans the width of the membrane, creating a pore. The cleavage of peptide bonds by OmpT is also dependent on the presence of bound lipopolysaccharide (LPS). When LPS is not present, the peptide binds too deeply within the active site, and the water cannot reach the carbonyl for its nucleophilic attack of the scissile bond.

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Part:BBa_K3000030

Normally this Lanthionine-containing peptide antibiotic (lantibiotic) active on Gram-positive bacteria. The bactericidal activity of lantibiotics is based on the depolarization of energized bacterial cytoplasmic membranes, initiated by the formation of aqueous transmembrane pores. In our project we have used this as an antimicrobial peptide to kill all the bacterial contaminants present in the media.

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Part:BBa_K3000027

These two in combination will help in releasing the bactericidal protein Subtilosin-A. Sbo-A has bactericidal activity against some gram-positive bacteria and some gram-negative bacteria. Sbo-A gene is used in combination with the alb-A gene to help in proper production and activation of Subtilosin-A protein. As the gene alb-A catalyzes the formation of 3 thioether bonds during the production of the sactipeptide subtilosin from SboA. In vitro the thioether bonds cannot be made in the absence of the SboA propeptide, suggesting this is the first reaction in subtilosin maturation.

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Part:BBa_K3000028

The gene skfA produces a 26-residue extracellular sporulation filling factor (SKF) that induces the lysis of other B.subtilis cells that have not entered the sporulation pathway. Used in our system to inhibit the spore bearer and to denature them.

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BBa_K3000001

Subparts:

BBa_K3000020- CAP Binding Site
BBa_K3000021- Lac promoter
BBa_K3000022- Lac operator
BBa_B0034- RBS
BBa_K3000030- nisin-A coding gene
BBa_K3000023- 6xHis Tag
BBa_K3000025- T7 terminator

Length: 365

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BBa_K3000002

Subparts:

BBa_K3000020- CAP Binding Site
BBa_K3000021- Lac promoter
BBa_K3000022- Lac operator
BBa_B0034- RBS
BBa_K3000026- ompT
BBa_K3000030- nisin-A coding gene
BBa_K3000023- 6xHis Tag
BBa_K3000025- T7 terminator

Length: 492

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BBa_K3000004

Subparts:

BBa_K3000020- CAP Binding Site
BBa_K3000021- Lac promoter
BBa_K3000022- Lac operator
BBa_B0034- RBS
BBa_K3000027- Sbo-A with alb-A gene
BBa_K3000023- 6xHis Tag
BBa_K3000025- T7 terminator

Length: 1735

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BBa_K3000007

Subparts:

BBa_K3000020- CAP Binding Site
BBa_K3000021- Lac promoter
BBa_K3000022- Lac operator
BBa_B0034- RBS
BBa_K3000028- skf coding gene
BBa_K3000023- 6xHis Tag
BBa_K3000025- T7 terminator

Length: 427