Difference between revisions of "Part:BBa K2842680"
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===USAGE AND BIOLOGY===
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===Usage and Biology===
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====PROTEIN POLYMERISATION BY SPLIT INTEINS====
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====Protein Polymerisation by Split Inteins====
 
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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.   
 
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.   
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Intein Monomer 1 utilises a Npu-C and an AceL-TerL-N, which are complementary to the flanked inteins on Intein Monomer 2. The Npu-C split intein is the C-terminal domain fragment from a Nostoc punctiforme (Npu) dnaE gene. It possesses more than 98% trans-splicing efficiency, which is greater splicing activity than other species’ DnaE inteins (Iwai et al., 2006). On the other hand, the AceL-TerL-N, derived from phage genes discovered in antarctic permanently stratified saline lakes, is the smallest N-terminal split intein as it is composed of only 25 amino acids .(Thiel et al., 2014).
 
Intein Monomer 1 utilises a Npu-C and an AceL-TerL-N, which are complementary to the flanked inteins on Intein Monomer 2. The Npu-C split intein is the C-terminal domain fragment from a Nostoc punctiforme (Npu) dnaE gene. It possesses more than 98% trans-splicing efficiency, which is greater splicing activity than other species’ DnaE inteins (Iwai et al., 2006). On the other hand, the AceL-TerL-N, derived from phage genes discovered in antarctic permanently stratified saline lakes, is the smallest N-terminal split intein as it is composed of only 25 amino acids .(Thiel et al., 2014).
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====Reporter Protein====
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Intein Monomer 1 encodes the reporter protein, red fluorescent protein (RFP). RFPs derive from various coral species, and are utilised to emit orange and fluorescence under UV-light (Miyawaki et al., 2013). In particular, Intein Monomer 1 uses a highly engineered mutant RFP isolated from Discosoma striata (coral).
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[[File:T--UCL--bsaI digests IP RFP GFP.png|400px|thumb|left|
 
[[File:T--UCL--bsaI digests IP RFP GFP.png|400px|thumb|left|

Revision as of 01:30, 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: Protein trans-splicing

Inteins ligate their flanking sequences with a native peptide bond. These sequences can be either their native exteins or unrelated peptides or proteins. (Thiel et al., Angewandte Communications, 2014)

Intein Monomer 1 utilises a Npu-C and an AceL-TerL-N, which are complementary to the flanked inteins on Intein Monomer 2. The Npu-C split intein is the C-terminal domain fragment from a Nostoc punctiforme (Npu) dnaE gene. It possesses more than 98% trans-splicing efficiency, which is greater splicing activity than other species’ DnaE inteins (Iwai et al., 2006). On the other hand, the AceL-TerL-N, derived from phage genes discovered in antarctic permanently stratified saline lakes, is the smallest N-terminal split intein as it is composed of only 25 amino acids .(Thiel et al., 2014).


Reporter Protein

Intein Monomer 1 encodes the reporter protein, red fluorescent protein (RFP). RFPs derive from various coral species, and are utilised to emit orange and fluorescence under UV-light (Miyawaki et al., 2013). In particular, Intein Monomer 1 uses a highly engineered mutant RFP isolated from Discosoma striata (coral).


Figure xxx: 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. coliB. subtilisS. cerevisiaeA. thalianaP. patensMammals
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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