Part:BBa_K2842680

Intein Monomer 1: RFP reporter flanked with orthogonal inteins

Intein Monomer 1
Function	Create intein-spliced polymers
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 [1]. 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 as depicted in Figure 1, 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 [2].

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 [3]. In particular, Intein Monomer 1 uses a highly engineered mutant RFP isolated from Discosoma striata (coral).

Related Entries in the Registry

The 2018 UCL team utilised the previously existing T7 promoter (BBa_J64997), Strep-tag (BBa_K823038), and terminator (BBa_B1006) within the Intein Monomer 1 design. This part works together with Intein Monomer 2 (BBa_K2842690), which was submitted by the 2018 UCL iGEM team.

New Part Status

Other than these specified registered basic parts, the rest of our Intein Monomer 1 construct is made up of newly submitted basic parts by the 2018 UCL iGEM team, therefore potentially making Intein Monomer 1 a novel biobrick. Intein Monomer 1 is also the only Biobrick registered with a AceL-TerL-C intein.

Usage

Polymerisation

Purified Intein Monomer 1 can react with Purified Intein Monomer 2 to form long polymers because of the orthogonal inteins that are complementary to each other. This polymerisation reaction occurs optimally at the combined reaction temperatures of both split intein species, which are 8°C for AceL-TerL intein and 37°C for Npu intein (Thiel et al., 2014) [4].

Functionalisation

Functional proteins can be Gibson Assembled into Intein Monomer 1. SapI restriction enzymes are used to remove the mRFP1, which creates GGC and GAG overhangs compatible with our Gibson overhangs 5’[GGCGTTGTTTCGCATAACTGTTATAAT]3’ and 5’[TGGAGCCACCCGCAGTTCGAAAAA]3’.

Primer Design

Forward Primer 5’[GCTTCTACAAACGCGGCTTCTT]3’
Reverse Primer 5’[ACGACGCCGgtTACTACATTGA]3’

These are the primers the 2018 UCL team used to PCR amplify and sequence the overall Intein Monomer 1 construct.

Forward Primer 5’[AACGGGTTCATCGCTAGTAATTGTTATAAT]3’
Reverse Primer 5’[AGTGGATCATGGAGCCACCCG]3’

These are the primers the 2018 UCL team used to PCR amplify BBa_K2842681, as well as during colony PCR analysis of any inserted protein of interest.

Figure 2: 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

Experimental Approach

DNA Analysis

To confirm the part sequence size, the 2018 UCL team ran BsaI digested samples to cut out Intein Monomer 1 out of the pSB1C3 backbone. This band is shown in Figure 2.1 at around 1200 bp.

Protein Analysis

The 2018 UCL team purified Intein Monomer 1 using a Strep-tag column. We then ran our sample on a SDS page gel against a PageRuler Plus marker, as shown in Figure 3.

Figure 3: SDS Page gel

(1) Purified BBa_K2842680 grown in LB media
(2) Purified BBa_K2842680 grown in TB media
*edited to show relevant bands

Fluorescence Analysis

Table 1. Fluorescence Measurements
Temperature	25°C	30°C	37°C
Grown in LB medium	600100	470790	271950
Grown in TB medium	241545	98385	361680

The 2018 UCL team transformed Intein Monomer 1 into One Shot™ BL21 Star™ (DE3) Chemically Competent E. coli. The cells were cultured in 50mL LB growth medium at 25°C, 30°C and 37°C degrees, until OD600 reached 0.6 when 400μM IPTG was added to induce expression of mRFP1 from the T7 promoter. Fluorescence was then measured after 17 hours of induction using the BMG FLUOstar Omega plate reader. Results are shown in Table 1.

Large Scale Protein Production

To scale-up production of Intein Monomer 1, the 2018 UCL team grew up cultures in 500mL shake flasks and analysed the growth at two temperatures, 25°C and 37°C. The scale-up workflow is documented in Table 2, and the results are depicted in Figures 4 and 5.

Table 2. Scale-up Workflow
Day 1 (evening)	Day 2 (morning)	Day 2 (evening)	Day 3 (morning)
transform 1 µL plasmid DNA in 30 µL competent cells (BL21), plate on antibiotics and leave in 37 °C condition overnight autoclave LB/medium	for each strain, prepare 8 x 10 ml LB starter cultures in 50 ml Falcon tubes by adding the appropriate antibiotics 10 µL Chloramphenicol is needed for 10ml LB starter.	pick several colonies from the LB agar plate using a sterile loop, inoculate in the starter cultures incubate the starter cultures overnight at 37 °C, 200 rpm autoclave the 250 ml baffled-flasks containing 50 ml of each medium	measure OD600 of overnight cultures

Figure 4

Figure 5

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.
The BioBrick-AutoAnnotator was created by TU-Munich 2013 iGEM team. For more information please see the documentation. If you have any questions, comments or suggestions, please leave us a comment.

References

[1] Protein Engineering Handbook. Chapter 10: Intein in protein engineering. Gillies AR and Wood DW. 2009 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim. Edited by Lutz S and Bornscheuer UT. Pages 271-294.
[2] Thiel IV, Volkmann G, Pietrokovski S and Mootz HD. (2014) An atypical naturally split intein engineered for highly efficient protein labeling. Angewandte Communications, Int. Ed., 53: 1306-1310.
[3] Miyawaki A, Shcherbakiva DM and Verkhusha VV. (2013) Red fluorescent proteins: chromophore formation and cellular applications. Curr Opin Struct Biol, 22: 679-688.
[4] Iwai H, Zuger S, Jin J and Tam PH. (2006) Highly efficient protein trans-splicing by a naturally split DnaE intein from Nostoc punctiforme. FEBS Letters, 580: 1853-1858.