Reporter

Part:BBa_K2814006

Designed by: Abhilash Patel   Group: iGEM18_IIT_Delhi   (2018-10-10)


pLTL_RBS+mKate2+TT

A reporter sequence containing hybrid promoter(lac-tet-lac) with bright fluorescent mKate2 gene along with a ssrA degradation tag and a terminator, a RBS preceded this sequence so that the hybrid promoter could be used in tandem with this reporter. Can be induced by IPTG or/and aTc depending upon the repressors acting upon it.

The pLTL (Lac-Tet-Lac) (BBa_K2814001) is a hybrid promoter. It is composed of the operator and promoter sequences of pTetand pLac promoter. There are multiple benefits of using the pLTL promoter:-

1) The hybrid pLTL shows almost complete repression on being repressed and on complete induction (either by IPTG or aTc), the hybrid pLTL promoter shows a 1.4-2 times higher expression.

2) The hybrid pLTL promoter also permits flexible gene expression because it can be utilized under either or both repression controls (LacI and TetR) simultaneously.

The RBS used is standard Elowitz RBS and we have use a codon optimized variant of mKate2 along with a ssrA degradation tag in order to speed up the degradation rate of the mKate2. More information related to the mKate2+ssrA degtag and the part sequence can be found on it’s BioBrick page (BBa_K2814009).The terminator used is a standard double terminator composed of rrnb T1 and T7 Te terminators. (BBa_K2814008)


Usage and Biology

mKate2 is characterized by complete and fast chromophore maturation at 37°C with maturation half-time <20 min (versus 40 min for mCherry). The high brightness, far-red emission spectrum, excellent pH resistance and photostability, coupled with low toxicity demonstrated in transgenic Xenopus laevis embryos, make mKate2 a superior fluorescent tag for imaging in living tissues.

mKate2 is mainly intended for protein labeling. Its far-red fluorescence allows easy and reliable separation from standard green fluorescent labels in dual-color high-throughput assays.


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI.rc site found at 562
    Illegal BsaI.rc site found at 751


Functional Parameters

Molecular weight, kDa 26
Polypeptide length, aa 232
Fluorescence color far-red
Excitation maximum, nm 588
Emission maximum, nm 633
Quantum yield 0.40
Extinction coefficient, M-1cm-1 62 500
Brightness* 25.0
Brightness, % of EGFP 74
pKa 5.4
Structure monomer
Aggregation no
Maturation half-time, min <20
Photostability, widefield** 69
Photostability, confocal** 390

Reference : Shcherbo D, Murphy CS, Ermakova GV, Solovieva EA, Chepurnykh TV, Shcheglov AS, Verkhusha VV, Pletnev VZ, Hazelwood KL, Roche PM, Lukyanov S, Zaraisky AG, Davidson MW, Chudakov DM. Far-red fluorescent tags for protein imaging in living tissues. Biochem J. 2009; 418 (3):567-74. doi: 10.1042/BJ20081949 / pmid: 19143658

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