Difference between revisions of "Team:IIT Delhi/Basic Part"

The pLTL (Lac-Tet-Lac) is a hybrid promoter. It is a promoter composed of the operator sequences of pTet and the pLac promoter. There are multiple benefits of using the pLTL promoter.

The hybrid pLTL shows almost complete repression on being repressed, and on induction (by IPTG and/or aTc), the hybrid pLTL promoter shows a 1.4-2 times higher expression.
The hybrid pLTL promoter also permits flexible gene expression because it can be utilized under either or both repression controls (LacI and TetR) simultaneously.

BBa_K2814008 = rrnB T1 Terminator + T7Te Terminator

Our part is double terminator composed of rrnB T1 Terminator(BBa B0010) and T7 Te Terminator(BBa B0012). Both of these are forward terminators that are extensively used in E. coli.

BBa_K2814009 = mKate2

Mkate2 - mKate2 is a red fluorescent protein derived from Entacmaea quadricolor. It possesses fluorescence with excitation maxima at 588 nm and emission maxima at 588 and 633 nm, mKate2 is almost 3-fold brighter than mKate. mKate2 can be used in labelling applications along with blue, cyan, green, yellow, and red fluorescent dyes. Its high pH-stability with pKa=5.4 makes it useful for imaging in acidic organelles, such as late and recycling endosomes and lysosomes.

BBa_K2814010 = rrnB T1 Terminator

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BBa_K2814007 = sfGFP_ssrA

Our part consists of sfGFP appended with a ssrA(LVA) deg-tag. It has been codon optimised for E. coli. This part is useful as it has high intensity as compared to other gfp variants as well as faster degradation rates and shorter reporter lifetimes.

Superfolder GFP is a basic (constitutively fluorescent) green fluorescent protein derived from Aequorea victoria. It has an emission wavelength of 510 nm and excitation wavelength of 485nm. It has a robust folding characteristic. The superfolder mutations also make the folding of GFP tolerant of mutations that would otherwise reduce the folding yield of GFP.

Our ssrA degtag is the gfp(LVA) that has a single (A → G) point mutation in nucleotide 349, resulting in an Asp117 → Gly117 (D117G) amino acid change. This point mutation does not appear to change the fluorescence spectrum of gfp(LVA). The LVA tag has been reported to lead to fast protein degradation, degrading GFP with rate -0.018 per minute. This corresponds to in vivo half-lives of mature Gfp(LVA) of approximately 40 min.

References

Pédelacq JD, Cabantous S, Tran T, Terwilliger TC, Waldo GS. Engineering and characterization of a superfolder green fluorescent protein. Nature biotechnology. 2006 Jan;24(1):79.
Andersen, J. B. et al. New unstable variants of green fluorescent protein for studies of transient gene expression in bacteria. Appl. Environ. Microbiol. 64, 2240–6 (1998).

BBa_K2814011 = attP TP901-1

AttP TP901-1 - It is the AttP site of TP901-1 integrase (serine based recombinase enzyme). TP901-1 integrase is an enzyme that has been isolated from TP901-1 phage. It is responsible for catalysing site-specific recombination at AttP and AttB sites. The recombination mechanism depends on the orientation of the AttP and AttB sites. These enzymes help the virus integrate its DNA into the bacterial genome. TP901-1 is widely used in the construction of logic gates and modification of DNA sequences.

BBa_K2814012 = BxbI Integrase + ssrA(LVA) deg tag

BxbI Integrase triggers attP × attB recombination. The product of attP × attB recombination is an integrated prophage flanked by two new recombination sites, attL and attR, each containing half sites derived from attP and attB. In the absence of accessory factors the integrases mediate unidirectional recombination between attP and attB with greater than 80% efficiency. In the presence of a phage-encoded accessory protein, the recombination directionality factor (RDF) the attP × attB recombination is inhibited and the attL × attR recombination is stimulated. Bxb1 integrase yields approximately two-fold more recombinants and displays about two fold less damage to the recombination sites than other phage-encoded serine integrases. Our BxbI Integrase has a ssrA deg tag attached to it for faster degradation rates.

BBa_K2814013 = attB TP901-1

AttB TP901-1 - It is the AttB site of TP901-1 integrase (serine based recombinase enzyme). TP901-1 integrase is an enzyme that has been isolated from TP901-1 phage. It is responsible for catalysing site-specific recombination at AttP and AttB sites. The recombination mechanism depends on the orientation of the AttP and AttB sites. These enzymes help the virus integrate its DNA into the bacterial genome. TP901-1 is widely used in the construction of logic gates and changing DNA sequence.

BBa_K2814014 = complement of B0034, RBS on the antisense strand, twin exists

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BBa_K2814015 = pLac(lambda) hybrid

pLac - Lambda hybrid - is a hybrid promoter consisting of Lac and Lambda operator sites in the core region. This hybrid promoter can be induced in the presence of IPTG or

BBa_K2814017 = attP Bxb1

AttP Bxb1 - It is the AttP site of Bxb1 integrase (Serine based recombinase). Bxb1 integrase carries out site-specific recombination by catalysing unidirectional recombination to produce AttL and AttR sites from AttP and AttB. This switching capacity allows them to be used in the design of toggle switches.

BBa_K2814018 = attB Bxb1

AttB Bxb1 - It is the AttB site of Bxb1 integrase (Serine based recombinase). Bxb1 integrase carries out site-specific recombination by catalysing unidirectional recombination to produce AttL and AttR sites from AttP and AttB. This switching capacity allows them to be used in the design of logic gates.

BBa_K2814019 = P7 Promoter

Constitutive P7 promoter - It is the complement of the constitutive P7 promoter so as to initiate transcription in the reverse direction.

BBa_K2814021 = BxbI-Xis + ssrA(LVA) deg tag------------

Xis-BxbI_ssrA deg tag - Consists of bxb1 excisionase followed by ssrA degradation tag. Bxb1-Xis catalyses the conversion of AttL and AttR sites to AttP and AttB sites when expressed with Bxb1 integrase, thereby reverting the recombination caused by integrase. ssrA deg tag degrades the Bxb1-Xis formed as an increase in the amount of excisionase renders the system inefficient. This property allows Bxb1 to be used in the construction of logic gates.

BBa_K2814022 = attB BxbI (reverse orientation)

AttB-BxbI (Reverse Orientation) - Contains AttB site of Bxb1 integrase in the reverse orientation (Reverse of BBa_K2814018). Bxb1 integrase carries out site-specific recombination by catalysing unidirectional recombination to produce AttL and AttR sites from AttP and AttB. This switching capacity allows them to be used in the design of logic gates.

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Undergraduate Laboratory
Department of Biotechnology and Biochemical Engineering, IIT Delhi

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