Difference between revisions of "Part:BBa K2065001"
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Latest revision as of 16:31, 19 October 2016
CT52-mNeonGreen
This BioBrick is a coding sequence for the CT52 protein, attached to the mNeonGreen protein with a GGS10 linker in between. Because CT52 has a binding interaction for the T14-3-3 scaffold protein under influence of the small molecule fusicoccin, it can be used for assembly of proteins on this scaffold protein. mNeonGreen is a fluorescent protein and together with CT52-SmallBit and CT52-LargeBit it can be used for a BRET assay on a tetrameric T14-3-3 scaffold protein.
Design of the construct
This parts is constructed of three subparts; CT52, a GGS10 linker and the fluorescent protein mNeonGreen. It has a molecular weight of 38 kDa. CT52 is the name for the isolated last 52 C-terminal amino acids of the plant plasma H+-ATPase PMA2 [1]. The N-terminus of CT52 is connected to a flexible GGS10 linker. This linker consists of a ten times repeating Glycine-Glycine-Serine amino acid sequence. mNeonGreen is a yellow-green fluorescent protein derived from a fluorescent protein from the Cephalochordate Branchiostoma Lanceolatum. For a fluorescence measurement, the mNeonGreen fluorophore can be excitated with a laser with a wavelength of 480 nm and read out at 517 nm. [2]
Usage
These three subparts were inserted in a pET28a vector for protein expression, and subsequently used for an in vitro BRET assay. This BRET assay tests the ability of the tetrameric variant of the T14-3-3 scaffold protein to assemble four different proteins.
Because the free C-terminus of CT52 has a strong binding affinity with the T14-3-3 scaffold from the Nicotiana plumbaginifolia (Tobacco plant) and the N-terminus can be bound to another protein, CT52 was used to test the activity of the heterotetrameric T14-3-3 scaffold protein. This heterotetrameric scaffold can bind four different proteins at the same time. Therefore, three mutation sets were designed (with the Rosetta Software package) to create orthogonal bindings between T14-3-3 and CT52. The fourth binding pocket is the wildtype pocket. For more information about the designing of the mutation sets for these new binding pockets, visit our wiki (link naar het Rosetta stuk)
For this BRET assay, the CT52-mNeongreen as well as CT52-LargeBit and CT52-SmallBit were assembled on the T14-3-3 scaffold protein. CT52-mNeonGreen will be the acceptor that changes the wavelength of the light emitted by NanoLuc to a wavelength of 517 nm. This system is visible in figure 1.
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Figure 1: Schematic representaton of the BRET system with tetrameric T14-3-3 scaffold protein and the NanoBiT system linked to CT52
Sequence
The sequence of our CT52-mNeonGreen has been verified by StarSeq. It contains the prefix and suffix with the correct restriction sites (EcoRI, XbaI, SpeI and PstI). CT52-mNeonGreen is 956 bp long.
Figure 2: Snapgene map of BBa_K2065001
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BamHI site found at 964
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
Fluorescence confirmation
The construct on which CT52-mNeonGreen was planned to be tested was created on DNA level but due to time limitations this construct was not expressed. This means mNeonGreen-CT52 has not been characterized yet.
mNeonGreen-CT52
| Protein Specifications | |||
|---|---|---|---|
| General Information | Number of amino acids | 344 | |
| Molecular weight | 37684 | ||
| Theoretical pi | 5.23 | ||
| Extinction coefficient | 27555 | ||
| Formula | C1643H2521N449O544S13 | ||
| Total numbers of atons | 45840 | ||
| Amino Acid Composition | Amino Acid | Frequency | Percentage(%) |
| Ala(A) | 18 | 5.2 | |
| Arg(R | 1 | 3.2 | |
| Asn(N) | 15 | 3.3 | |
| Asp(D) | 15 | 3.3 | |
| Cys(C) | 1 | 0.3 | |
| Gln(Q) | 13 | 3.8 | |
| Glu(E) | 31 | 9.0 | |
| Gly(G) | 44 | 12.8 | |
| His(H) | 9 | 2.6 | |
| Ile(I) | 11 | 3.2 | |
| Leu(L) | 20 | 5.8 | |
| Lys(K) | 22 | 6.4 | |
| Met(M) | 12 | 3.5 | |
| Phe(F) | 13 | 3.8 | |
| Pro(P) | 12 | 3.5 | |
| Ser(S) | 27 | 7.8 | |
| Thr(T) | 36 | 10.5 | |
| Trp(W) | 4 | 1.2 | |
| Tyr(T) | 16 | 4.7 | |
| Val(V) | 14 | 4.1 | |
| Pyl(O) | 0 | 0.0 | |
| Sec(U) | 0 | 0.0 | |
References
[1] - Svennelid, F., Olsson, A., Piotrowski, M., Rosenquist, M., Ottman, C., Larsson, C.& Sommarin, M. (1999). Phosphorylation of Thr-948 at the C terminus of the plasma membrane H+-ATPase creates a binding site for the regulatory 14-3-3 protein. The Plant Cell, 11(12), 2379-2391.
[2] - N.C. Shaner et al,͞A bright monomeric green fluorescent protein derived from Branchiostoma lanceolatum.,Nature Methods, vol. 10, no. 5, pp. 407-409, Mar. 2013.