Difference between revisions of "Part:BBa K2842680"

Revision as of 01:18, 18 October 2018

Intein Monomer 1: RFP reporter flanked with orthogonal inteins

Intein Monomer 1
Function	Standardised blue-white screening
Use in	E. coli cells
Chassis Tested	DH5α cells, BL21* cells
Abstraction Hierarchy	Composite Device
Related Device	BBa_K2842690
RFC standard	RFC10,RFC12,RFC21,RFC23 & RFC25 compatible
Backbone	pSB1C3
Submitted by	[http://2018.igem.org/Team:UCL UCL iGEM 2018]

This gene encodes a novel split-intein flanked reporter device which enables the use of intein splicing for any protein of interest through SapI digestion. Intein Monomer 1 was created to work in conjunction with its complimentary composite part Intein Monomer 2 to construct a intein polymerisation system.

USAGE AND BIOLOGY

PROTEIN POLYMERISATION BY SPLIT INTEINS

Discovered in the late 1980s, inteins are naturally occurring protein segments attached to specific host proteins of unicellular organisms (Protein Engineering Handbook, 2009). Inteins contain both an N- and C-terminal domain, which can be split to allow either half to be bound to unique external proteins. Matching split inteins self-excise from their attached host protein in a trans-splicing reaction (depicted in Figure X), which allows for the ligation of the external proteins through a peptide bond.

Figure 1: BsaI digestions

(1) BsaI digested Intein Passenger BBa_K2842669
(2) BsaI digested RFP inteins BBa_K2842680
(3) BsaI digested GFP inteins BBa_K2842690
*edited to show relevant bands

Sequence and Features

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 site found at 1165
Illegal BsaI.rc site found at 28
Illegal SapI site found at 903
Illegal SapI.rc site found at 213

Functional Parameters

Protein data table for BioBrick BBa_ automatically created by the BioBrick-AutoAnnotator version 1.0

Nucleotide sequence in RFC 10: (underlined part encodes the protein)
GCTTCTACAAACGCGGCTTCTTCCAAAGAGACCTAATACGACTCACTATAGGGGTTGTGAGCGGATAACAACCCAAGACAAGGAGGAGTACCAATGATCAAG ... CGCTTGGCT
TAAGTGACAGTTGAAAAGCGAAAAAAAAACCCCGCCCCTGACAGGGCGGGGTTTTTTTTGGTCTCAACGGACGACGCCGGTTACTACATTGA
ORF from nucleotide position 94 to 1032 (excluding stop-codon)

Amino acid sequence: (RFC 25 scars in shown in bold, other sequence features underlined; both given below)

1
101
201
301

MIKIATRKYLGKQNVYDIGVERDHNFALKNGFIASNCYNGGRASMASSEDVIKEFMRFKVRMEGSVNGHEFEIEGEGEGRPYEGTQTAKLKVTKGGPLPF
AWDILSPQFQYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFEDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASTERMYPEDGA
LKGEIKMRLKLKDGGHYDAEVKTTYMAKKPVQLPGAYKTDIKLDITSHNEDYTIVEQYERAEGRHSTGAGSSSESGSWSHPQFEKAEYCVYGDTMVETED
GKIKIEDLYKRLA*

Sequence features: (with their position in the amino acid sequence, see the list of supported features)

Strep-tag II:

278 to 285

Amino acid composition:

Ala (A)	18 (5.8%)
Arg (R)	13 (4.2%)
Asn (N)	9 (2.9%)
Asp (D)	19 (6.1%)

Cys (C)	2 (0.6%)
Gln (Q)	10 (3.2%)
Glu (E)	29 (9.3%)
Gly (G)	32 (10.2%)

His (H)	7 (2.2%)
Ile (I)	15 (4.8%)
Leu (L)	16 (5.1%)
Lys (K)	30 (9.6%)

Met (M)	11 (3.5%)
Phe (F)	13 (4.2%)
Pro (P)	13 (4.2%)
Ser (S)	20 (6.4%)

Thr (T)	17 (5.4%)
Trp (W)	4 (1.3%)
Tyr (Y)	17 (5.4%)
Val (V)	18 (5.8%)

Amino acid counting

	Total number:	313
	Positively charged (Arg+Lys):	43 (13.7%)
	Negatively charged (Asp+Glu):	48 (15.3%)
	Aromatic (Phe+His+Try+Tyr):	41 (13.1%)

Biochemical parameters

	Atomic composition:	C₁₅₆₉H₂₄₁₇N₄₁₉O₄₈₃S₁₃
	Molecular mass [Da]:	35294.7
	Theoretical pI:	5.90
	Extinction coefficient at 280 nm [M^-1 cm^-1]:	47330 / 47455 (all Cys red/ox)

Plot for hydrophobicity, charge, predicted secondary structure, solvent accessability, transmembrane helices and disulfid bridges

Codon usage

	Organism:	E. coli	B. subtilis	S. cerevisiae	A. thaliana	P. patens	Mammals
	Codon quality (CAI):	good (0.80)	good (0.72)	good (0.67)	good (0.75)	good (0.79)	good (0.71)

Alignments (obtained from PredictProtein.org)
There were no alignments for this protein in the data base. The BLAST search was initialized and should be ready in a few hours.

Predictions (obtained from PredictProtein.org)

There were no predictions for this protein in the data base. The prediction was initialized and should be ready in a few hours.

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References

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@@ Line 35: / Line 35: @@
 </p>
+===USAGE AND BIOLOGY===
+====PROTEIN POLYMERISATION BY SPLIT INTEINS====
+<p>
+Discovered in the late 1980s, inteins are naturally occurring protein segments attached to specific host proteins of unicellular organisms (Protein Engineering Handbook, 2009). Inteins contain both an N- and C-terminal domain, which can be split to allow either half to be bound to unique external proteins. Matching split inteins self-excise from their attached host protein in a trans-splicing reaction (depicted in Figure X), which allows for the ligation of the external proteins through a peptide bond.
+</p>