View source for Part:BBa K4674001


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<partinfo>BBa_K4674001 short</partinfo>

This part is modified from the BBa_K4674000 (mGL). The C-terminal of BBaK467000 (mGL) is fused with a linker composed of 4 alanines, following a C7 dodecapeptide peptide for folate receptor alpha recognization. 

The mGL fluorescent protein is developed for the expression in the mammalian systems, since the low soluibity or misfolding of eGFP limit the total fluorescence output of whole cells.

<strong> Cellular Brightness and Maturation </strong> 

In the characterization, nGL shows 630% greater brightness than eGFP. The maturation half-time of mGL is 13.5 min, which is 207% faster than eGFP (33.0min). 
mGL is compatible with common EGFP filter sets and the popular 488-nm argon-ion laser line for widefield and confocal microscopy. Its excitation and emission peaks of 503/514 nm additionally make it well matched to newer solid-state 491-nm lasers and 505-nm light-emitting diodes (LEDs). 

<strong> Biochemical Characterization </strong> 

The QY and EC of mGreenLantern (ϕ = 0.72, ε = 101 mM−1⋅cm−1) compare favorably with those of other bright GFPs such as Clover (ϕ = 0.75, ε = 97 mM−1⋅cm−1), and its low pKa of 5.6 indicates greater acid resistance than EGFP.

mGreenLantern completely lacks a 405-nm absorbance peak, unlike EGFP and Clover, suggesting that it will be better suited for multicolor imaging with T-Sapphire.

<strong> Chemical and thermal stability </strong> 
In the strongly denaturing solution of 6.3 M guanidinium HCl, pH 7.5, mGL persisted for 100 s before reaching half the fluorescence of its native control wells, while other fluorescent protein (e.g. eGFP, eYFG, and Clover) denatured instantly. The melting temperture of mGL is 87.2 °C, which is much higher than the mNeonGreen (68.0 °C), eGFP (80.3 °C), or sfGFP (86.4 °C)

<strong> Antibody recognition </strong> 
The mGL protien could be recognized at least by three commercial antibodies (goat α-GFP polyclonal, Abcam, #ab6673; or goat α-GFP polyclonal, Novus, #NB1001770; or Ms α-GFP monoclonal, Life Technologies, #A-11122).  The truth that 91% sequence identity of mGL shared with eGFP strongly implies that mGL is readily compatible with existing technology for EGFP affinity-related applications.

<strong> Photostability </strong> 
When analyzed using normalized bleaching plots beginning at time 0 to include the Emerald-like behavior, mGreenLantern’s photostability was better than Venus and lower than Clover.
 

https://static.igem.wiki/teams/4674/wiki/mgl-table1-2.png "

Table 1.  Spectroscopic characterization of fluorescent protein (quoted from (Campbell et. al. 2020)


<strong>C7 dodecapeptide </strong> 
The sequence of C7 peptide is Met-His-Thr-Ala-Pro-Gly-Trp-Gly-Tyr-Arg-Leu-Ser, which is developed from the phage library screening. The ability of C7 peptide to recognize FRα is proved by FACS analysis of SKOV3 cell lines in vitro and the in vivo tumor staining. The equilibrium dissociation constant (KD) value between C7 and FRα was 0.3 μM. Importantly, the computational modeling and interaction analysis indicated that C7 binds with FRα close at the entrance of the pocket, which avoid the competition to folic acid (Xing et al. 2018)

<strong>The design of mGL-4A-C7 </strong> 
To efficiently label the CTCs captured by DNA tetrahedron, we first perform modeling and determine to link mGL protein and C7 dedocapeptide with 4 alaines (mGL-4A-C7). To expression the mGL-4A-C7 fusion protein, we design and cloned the composite par BBa_K4674010 into the pET15b. The fusion protein was induced by IPTG and purified by FPLC. Finally, to confirm whether the mGL-4A-C7 is functional, we perform the labeling experiments by SKOV3 cell line, which is the mimic of the captured CTC. (Due to the highly expression of FRα in SKOV3 cells)

https://static.igem.wiki/teams/4674/wiki/fig-9-design-okik-2.png

<strong> Reference </strong> 
Benjamin C. Campbell, Elisa M. Nabel, Mitchell H. Murdock, and Gregory A. Petsko.
mGreenLantern: a bright monomeric fluorescent protein with rapid expression and cell filling properties for neuronal imaging.
Proc Natl Acad Sci U S A. 2020 Dec 1;117(48):30710-30721

Lijun Xing, Yifeng Xu, Keyong Sun, Hong Wang, Fengguo Zhang, Zhengpin Zhou, Juan Zhang, Fang Zhang, Bilgen Caliskan, Zheng Qiu, Min Wang
Identification of a peptide for folate receptor alpha by phage display and its tumor targeting activity in ovary cancer xenograft
Sci Rep. 2018 May 30;8(1):8426.

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===Usage and Biology===

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<span class='h3bb'>Sequence and Features</span>
<partinfo>BBa_K4674001 SequenceAndFeatures</partinfo>


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===Functional Parameters===
<partinfo>BBa_K4674001 parameters</partinfo>
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