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Summary
Our best basic part is a novel split chloramphenicol resistance. The two split parts are BBa_K2926076 and BBa_K2926077, whereas BBa_K2926076 is the N-terminal part of the protein and BBa_K2926077 represents the C-terminal part. The resistance was split using trans-intein-splicing. Since chloramphenicol is the standard antibiotic resistance used by the iGEM community, our part will be beneficial for all future teams. Additionally we would like to introduce you to our basic parts, which are summarized at the bottom of the page.

	Chloramphenicol
	Intein-mediated protein splicing
	Split selection markers
	Biosafety
	Model and plasmid design
	Characterization

Chloramphenicol

Chloramphenicol is an antibiotic which easily diffuses across cell membranes. Once inside the cell it interferes in the protein biosynthesis pathway. More precisely it binds to specific residues of the 23S rRNA of the 50S ribosomal subunit and interrupts the formation of new peptide bonds.
In 1947 chloramphenicol was first discovered in an isolate from Streptomyces venezuelae, later that decade it became the first artificially produced antibiotic. (Pong 1979) In every day life chloramphenicol serves as a back up broadband antibiotic which is used in case other antibiotics can not be applied. Normally it is only used in case of need, since it comes with a broad range of side effects. Nevertheless, chloramphenicol is often one of the ingredients in eye ointments for the treatment of conjunctivitis. (Edwards 2009) Furthermore it is used to treat hazardous bacterial infections like plaque, cholera, MRSA or typhoid fever. (Ingebrigtsen 2017)
Additionally, it is one of the most commonly used antibiotics in molecular biology for the selection of genetically modified organisms. Many of the vectors for gene transfer used in research carry a gene encoding a chloramphenicol resistance. Thus, if a cell has taken up the vector which is encoding the gene of interest as well as the resistance gene this cell becomes resistant against chloramphenicol.

Resistance

Ribbon structure of the chloramphenicol acetyltransferase chloramphenicol bound to the catalytic center. (From PDB: 3CLA)(Leslie, A.G.W. 1990)

The choloramphenicol acetyltransferase (CAT) is an enzyme originally identified in Escherichia coli, that mediates resistance to chloramphenicol. (Shaw 1983) CAT covalently attaches an acetyl group from acetyl-CoA to chloramphenicol, rendering the acetylated chloramphenicol unable to bind to the 23S rRNA. (Shaw 1991) It is encoded by the cat gene which is commonly used by the iGEM community as part BBa_J31005 and in the standard backbone pSB1C3.

Intein-mediated protein splicing.

Inteins

An intein is a segment of a precursor protein which is able to mediate its own excision form the precursor protein, while joining the flanking protein sequences together, creating a new peptide bond.
The intein-mediated protein splicing occurs after the mRNA is translated into a sequence of amino acids. Prior to the splicing process the N-terminal part of the precursor protein is called N-extein, the center part is the intein and the C-terminal part is named C-extein. The spliced protein is called an extein as well.

Split inteins

A very special but small subset of inteins are the so-called split-inteins. They are transcribed and translated as separate polypeptides but rapidly associate afterwards to form the active intein. The active intein then ligates the fused N- and C-exteins in a process called protein trans-splicing.

Mechanism of split intein-mediated protein splicing. The intein autocatalytically cleaves itself out of the polypeptide, connecting both adjacent exteins by a peptide bond.

After the first split intein SspDnaE, a subunit of the DNA polymerase III (DnaE) from Synechocystis sp. strain PCC6803 was discovered. Several homologous inteins were identified in cyanobacteria. (Wei et al. 2006, Dassa et al. 2007) However apart from sequence analysis there have only been few attempts to further characterize or compare their splicing efficiencies. (Dassa et al. 2007) Nevertheless a split intein from the cyanobacterium Nostoc punctiforme (NpuDnaE) which is highly homologous to Ssp DnaE was analyzed quite extensively. It has shown higher activities than SspDnaE in vivo and in vitro. (Iwai et al. 2006; Zettler et al. 2009). Additionally it has a boarder range of acceptance when it comes to the residues at the end of the C-extein. (Iwai et al. 2006).

Amino acids neighboring split sites

NMR solution structure of NpuDnaE (PDB code: 2KEQ) (Oeemig et al. 2009)

As implied, the activity of most split inteins is strongly dependent on the residues of the amino acids at the transition points between the intein and the extein. Therefore, the number of ligation sites for a natural split intein within a target protein is limited to sequences similar to endogenous extein sequences. The intein autocatalytically cleaves itself out of the polypeptide, connecting both adjacent exteins by a peptide bond. It has been shown that some amino acids are more favorable in the positions neighboring the splicing site than others. Especially the three amino acids right before (in the N-extein) and right after (in the C-extein) the splicing site are crucial. Not only the likelihood of correct splicing but also the speed of the splicing reaction are strongly depending on these amino acids. In almost all cases cytosin must be in the +1 position of the C-extein. For more information about the other amino acids possible in the crucial positions have a look at our Model page.

The highlighted three amino acids in the exteins bordering on the inteins are the most relevant for a successful splicing process. These are the amino acids are the ones that are known to have the biggest impact on the functionality of the splicing process (Cheriyan, Pedamallu, Tori, & Perler, 2013).

Mechanism

For a better understanding of the importance of the amino acid residues near the splicing site, following is a short summary of the split-intein-mediated splicing mechanism:

Split inteins associate
Nucleophilic attack of the peptide bond between the N-extein and the intein by the residue of the first amino acid in the intein
Formation of a linear ester (not shown) or thioester intermediate
Side chain of the first residue of the C-extein attacks the thioester (or ester), freeing the N-terminal end of the intein
N- and C-extein are connected, but not yet by a peptide bond
Amide nitrogen atom from the residue of the last amino acid of the intein cleaves the peptide bond between the intein and the C-extein
Intein is free
Free amino group of the C-extein attacks thioester (or ester) linking the exteins together
O-N or S-N shift forms a peptide bond creating a fully functional protein

Split-resistance genes

Split inteins enable us to divide and fuse all kinds of proteins. Their application ranges from structural biology, especially NMR spectroscopy to split intein-mediated production of genetically encoded cyclic peptide libraries. (Horshill and Benkovic 2006; Volkmann and Iwai 2010) Another possible application are split markers, like antibiotics for the selection of GMOs.
Researchers working in the field of synthetic biology are often using two or multiple plasmid systems to incorporate all desired transgenes into one cell. For the selection of positive clones and stable retention of the plasmids they usually apply several antibiotics to the cultivation medium. Unfortunately, multiple antibiotics provide very harsh growth conditions for the cells.
Split antibiotics are a great solution to reduce the number of needed antibiotics. A scheme how split markers are implemented is illustrated in figure 8. To create a split selectable marker the gene encoding an antibiotic resistance is split into two segments. Each segment is fused to one part of a split intein which is able to join the protein segments back together. Each of these fusion segments is inserted into a different vector. If a cell is transformed with both cohesive vectors it is able to develop a resistance against the respective antibiotic via protein trans-splicing. If the cell has taken up only one of the vectors it has no chance to survive selection since it can produce only one part of the protein mediating the antibiotic resistance.

Using split selectable markers it is possible keep two vectors encoding genes of interest inside a cell while using only one instead of two antibiotics. Which is beneficial not only for the growth conditions of the cells but also for the financial aspects of a project, since antibiotics are relatively expensive.

Biosafety

When using split selectable markers one part of the protein is encoded by a plasmid whereas the other part could be encoded by either another plasmid or the genome of the target organism. In both cases this system would increase the biosafety of the systems encoded on the plasmids. If one of the plasmids, a strain containing only one of the plasmids, or a strain encoding one of the parts in its genome would be released into the environment the antibiotic resistance would not spread. Since both parts of the protein are needed to establish the resistance, the uptake of only one of the parts would not increase the fitness of an organism in the outer world. This reduces the probability of GMOs to overgrow natural populations in the environment. Therefore, this system could increase the safety of GMOs in any application outside of the lab. This would be beneficial for the future application of many iGEM projects.

Model and plasmid design

Preliminary considerations

To assemble our Troygenics we are working with a two plasmid system. Since most of the real world applications of the Troygenics involve their release into the outer world, the biosafety of our system is a crucial part of our project. As explained above split selectable markers are a great way to improve the biosafety of two plasmid systems, because they not only reduce the likelihood of spread antibiotic resistances but also reduce the probability of GMOs overgrowing naturally occurring populations. As chloramphenicol is the standard antibiotic resistance used by the iGEM community but was never submitted as a split antibiotic resistance before we decided to develop a split chloramphenicol resistance, which is particular useful for future iGEM teams. It was our aim to split the resistance gene cat so that the two parts of the chloramphenicol acetytransferase are encoded by two different plasmids. According to the advantages mentioned above, we decided to use the split intein NpuDnaE (Partnummern)to create our kanamycin split resistance.
The important amino acids neighboring the native splice site of NpuDnaE are“Ala Glut Tyr” for the N-extein and “Cys Phe Asn” for the native C-extein. (Cheriyan et al. 2013) To examine the optimal split point of the chloramphenicol acetyltransferase which mediates the chloramphenicol resistance we designed and executed a model. It provides us with a list of all possible split points, ranked from best to worst, for the desired protein, based on its amino acid sequence and 3D structure.

Modeling the optimal split point

Our model takes the structure as well as the sequences around potential split points into account. At first split-points in regions which form an alpha-helix or a beta-sheet are avoided. Additionally the amino acids neighboring the splicing site are inspected. As previously shown, that certain amino acids are more favorable in these positions, leading to more correct splicing events as to an overall increased splicing rate. Based on previously reported data (Cheriyan, Pedamallu, Tori, & Perler, 2013), a score between 0 and 50 has been assigned to imply the value of an amino acid at a certain position. The higher the value the more favorable is the amino acid in that position for faster and more specific splicint. The positions relevant for this kind of splicing are the three ones right before and right after the insertion site for the split-intein (Fig. 1).

The modeling results indicate that the best split point would be after amino acid 30.

Relative favorability of each amino acid i (rF(A_i)) at each relevant position j neighboring the inteins. Adjusted from Cheriyan, Pedamallu, Tori, & Perler, 2013. The amino acids neighboring a splicing site were randomly mutated, +1 was set to Cysteine. The mutants conducting successful protein splicing were selected and the 6 neighboring amino acids were determined. To obtain the rf(A_i) values the occurence of each amino acid in each position was counted and this number was divided by the natural frequency of said amino acid. For our model Cysteine +1 was set to “50” to ensure that this was picked over any other combinations. Additionally, Methionine +2 was set to 20, as other sources stated that it might be an appropriate substitute for Cysteine (Brenzel, 2009).
Amino Acid	rF(A_i) at the N-terminal extein			rF(A_i) at the C-terminal extein
	-3	-2	-1	+1	+2	+3
D	1.39	1.52	0.00	0.00	0.00	1.26
E	1.26	3.51	0.07	0.00	0.00	5.5
N	0.93	1.26	2.19	0.00	0.00	8.61
Q	1.39	0.86	0.33	0.00	0.00	0.27
H	1.06	1.26	2.19	0.00	0.00	8.61
K	1.59	0.93	4.44	0.00	0.07	0.00
R	1.81	0.2	0.86	0.00	0.00	0.00
S	0.99	1.24	1.37	0.00	0.00	0.07
C	0.46	0.40	0.80	50	0.07	0.07
T	0.63	0.89	1.59	0.00	0.00	0.96
P	0.80	1.52	0.00	0.00	0.00	0.00
G	4.67	2.68	0.17	0.00	0.00	0.03
A	0.63	0.83	1.92	0.00	0.00	0.00
V	0.56	0.50	0.23	0.00	0.03	0.10
I	0.07	0.53	0.00	0.00	0.00	0.40
L	0.11	0.57	0.75	0.00	0.00	0.82
M	0.13	0.73	1.26	20	0.07	2.25
F	0.13	0.73	1.26	0.00	0.07	2.25
Y	0.20	0.60	2.05	0.00	0.13	5.23
W	0.07	0.40	0.99	0.00	29.42	0.07

To identify the optimal split point for intein-mediated splicing of proteins, all possible 6 amino acid long fragments of the peptide sequences were assessed by a python script. The sum of their rf(A_i)_j values was calculated for each fragment, revealing how well the center of the respective fragment would act as a split point.

The generation of all possible 6 amino acid long fragments of the peptide sequence of interest.

**Formula used to calculate how beneficial a certain combination of amino acids is for Npu DnaE mediated protein splicing.**

Using this approach, a list with all possible split points sorted by B(seq)_n is created, ranging from the best to the worst possible split point in the protein.
Complementing the sequence based determination of potential split points the desired structure of the final protein has to be considered. Positions in important structural features of the protein were determined using the protein structure viewer Chimera (Pettersen et al., 2004). Sequences involved in relevant structures were integrated in the analyses as described above to prevent the identification of possible split points in these regions. Lastly, the remaining list of split points was sorted from best to worst and the best split sequences determined were written FASTA files. Using the MODELLER-software (Sali & Webb, 1989), the most likely structure of the N- or C-terminal part of the protein fused to the split intein, prior to finding the other intein in the cell, was determined. To do so, it was necessary to find proteins with similar structures as templates for homology modeling. They were chosen as follows:

Model for the split chloramphenicol Resistance

We aimed to split the protein conveying resistance to chloramphenicol, chloramphenicol acetyltransferase (BBa_J31005). The resistance protein is commonly used within iGEM, thus making a split Chloramphenicol acetyltransferasa a vluable addition to the iGEM partsreg. The protein sequence of this part can be found here. According to our modeling, the best split point to use when introducing Npu DnaE was VAQ-CTY in the positions 28-33, the B(_VAQCTY) is 56.95.
Even though this split point is located at the second amino acid of a beta-sheet, we decided to test whether splitting at this position could lead to Chloramphenicol acetyltransferase fragments that could be reconstituted by intein-mediated protein splicing.
Similar to our modeling of the split kanamycin resistance, we used the homology modeling software MODELLER (Sali & Webb, 1989) to visualize each part of the chloramphenicol acetyltransferase fused to the Npu DnaE intein.

The amino acid sequences given to the software were:

Part 1:
MEKKITGYTTVDISQWHRKEHFEAFQSVAQCLSYETEILTVEYGLLPIGKIVEKRIECTVYSVDNNGNIYTQPVAQWHDRGEQEVFEYCLEDGSLIRATKDHKFMTVDGQMLPIDEIFERELDLMRVDNLPNNLEGHHHHHH

Part 2:
MIKIATRKYLGKQNVYDIGVERDHNFALKNGFIASNCCTYNQTVQLDITAFLKTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSSLWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANMDNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA

The templates used for homology modeling were:

Part 1	Part 2
1NOC, chain B (Chloramphenicol acetyltransferase)	1NOC, chain B (Chloramphenicol acetyltransferase)
4QFQ, chain A (Npu DnaE)	4QFQ, chain B (Npu DnaE)
5OL6, chain A (inactivated Npu SICLOPPS intein with CAFHPQ extein)	5OL6, chain B (inactivated Npu SICLOPPS intein with CAFHPQ extein)
4KL5, chain A (Npu DnaE)	1QCA, chain A (Type III Chloramphenicol acetyltransferase)
2KEQ, chain A (DnaE intein from Nostoc punctiforme)	3CLA, chain A (Type III Chloramphenicosl acetyltransferase)

After running the MODELLER software (Sali & Webb, 1989), two protein structures were calculated and placed adjacent to each other using Chimera (Pettersen et al., 2004).

The predicted 3D-structure of each subunit of the Cloramphenicol acetyltransferase split at the optimal predicted split point VAQ-CTY prior to splicing. It was developed using the homology modeling software MODELLER (Sali & Webb, 1989).

Plasmid design

We designed 4 parts encoding this chloramphenicol split resistance. Part BBa_2926076 and BBa_2926077 are basic parts. BBa_2926076 is encoding the N-terminal part of the split resistance, fused to the N-intein. This part was constructed in pSB3T5. Whereas BBa_2926077 encodes the C-intein fused to the C-terminal part of the split resistance and was constructed in pSB1K3.
Additionally to the split chloramphenicol resistance described on this page, we have constructed another chloramphenicol split resistance, as well as a kanamycin, ampicilin and a hygromycin split resistance. Here you can get more information on our handy collection of split antibiotic resistances.

Characterization

We have successfully constructed all parts and verified their nucleotide sequence with sanger sequencing.
After co-transformation of BBa_2926076 and BBa_2926077 into E. coli DH5α the cells where plated on selection plates containing kanamycin and tetracyclin, but no chloramphenicol since the cells need some time to establish the chloramphenicol resistance.
Afterwards a number of colonies were transferred to numerous plates, each containing a different concentration from 0.1 up to 100 μg/mL chloramphenicol. Alongside with our colonies containing the split chloramphenicol resistance we plated E. coli DH5α as a negativ control and E. coli DH5α containing pSB1C3, which is encoding RFP as a positive control.
We conducted this experiment as a triplicate. The results of one of the triplicates are depicted in figure 10. Native E. coli DH5α was only growing on the plates containing 0.1 μg/mL chloramphenicol, implying that the native E. coli DH5α has no significant resistance to chloramphenicol. E. coli DHα containing pSB1C3, which is encoding RFP was proven to be resistant on all tested chloramphenicol concentrations. The E. coli DH5α encoding our split chloramphenicol resistance showed to be resistant to chloramphenicol concentrations of averaged 7 μg/mL.
In every day lab life chloramphenicol is used in concentrations between 10 and 25 μg/mL. Since our split system shows resistance against chloramphenicol for concentrations of up to averaged 7 μg/mL the working concentrations for chloramphenicol could be reduced, leading to a diminished chloramphenicol consumption.

**Resistance test of split chloramphenicol resistance** with *E. coli DH5α* (in the top left section of every plate), *E. coli DH5α* containing pSB1C3_RFP (in the bottom section of every plate) and *E. coli DH5α* containing our chloramphenicol split resistance (in the top right section of every plate) plated on LB plates with of 0.1, 1, 7, 10, 20, 40, 60, 80, 100 μg/mL chloramphenicol (left to right and top to bottom)

List of our basic Parts

List of all basic parts
Basic parts	Description	Designer	Length
BBa_K2926000	Cas13a Lsh	Ina Schmitt, Isabel Conze, Katharina Wolff, Johanna Opgenoorth	4168 bp
BBa_K2926001	Cas13a Lbu	Ina Schmitt, Isabel Conze, Katharina Wolff, Johanna Opgenoorth	3480 bp
BBa_K2926003	GALL yeast promoter (validated part)	Ina Schmitt, Isabel Conze, Katharina Wolff, Johanna Opgenoorth	430 bp
BBa_K2926004	SNR52 yeast promoter	Ina Schmitt, Isabel Conze, Katharina Wolff, Johanna Opgenoorth	269 bp
BBa_K2926005	TPS1 yeast terminator	Ina Schmitt, Isabel Conze, Katharina Wolff, Johanna Opgenoorth	250 bp
BBa_K2926006	SUP4 yeast terminator	Ina Schmitt, Isabel Conze, Katharina Wolff, Johanna Opgenoorth	20 bp
BBa_K2926007	Inverted terminal repeat form S. cerevisiae gen ADE2, front	Ina Schmitt, Isabel Conze, Katharina Wolff, Johanna Opgenoorth	50 bp
BBa_K2926008	gRNA array (cas13a) with sgRNAs (for S.cerevisiae) gRNA1-3	Ina Schmitt, Isabel Conze, Katharina Wolff, Johanna Opgenoorth	187 bp
BBa_K2926009	gRNA array (cas13a) with sgRNAs (for S.cerevisiae) gRNA1-7	Ina Schmitt, Isabel Conze, Katharina Wolff, Johanna Opgenoorth	435 bp
BBa_K2926010	gRNA array (cas13a) with sgRNAs (for S.cerevisiae) gRNA4-7	Ina Schmitt, Isabel Conze, Katharina Wolff, Johanna Opgenoorth	301 bp
BBa_K2926011	gRNA array (cas13a) with 3 sgRNAs (for A. niger)	Ina Schmitt, Isabel Conze, Katharina Wolff, Johanna Opgenoorth	207 bp
BBa_K2926013	Inverted terminal repeat form S. cerevisiae gen ADE2, back	Ina Schmitt, Isabel Conze, Katharina Wolff, Johanna Opgenoorth	50 bp
BBa_K2926015	Aspergillus niger 5S rRNA	Ina Schmitt, Isabel Conze, Katharina Wolff, Johanna Opgenoorth	119 bp
BBa_K2926020	Promotor of protein VIII from the M13 phage	Astrid Többer, Nefeli Chanoutsi	47 bp
BBa_K2926021	sgRNA for the detection of RFP with Cas13a	Ina Schmitt, Isabel Conze, Katharina Wolff, Johanna Opgenoorth	57 bp
BBa_K2926022	Trp_sfGFP_Opy2p_mCherry_M13K07 gene VIII_M13K07 gene III	Johanna Opgenoorth	4066 bp
BBa_K2926023	M13K07_genes_II_VIII	Astrid Többer, Nefeli Chanoutsi	2015 bp
BBa_K2926025	M13K07_genes_III_IV	Astrid Többer, Nefeli Chanoutsi	2820 bp
BBa_K2926026	M13K07_truncated_III	Astrid Többer, Nefeli Chanoutsi	456 bp
BBa_K2926027	mCherry_VIII_fusion_protein	Astrid Többer, Nefeli Chanoutsi	1086 bp
BBa_K2926048	mCherry with hexahistidine tag for purification	Johanna Opgenoorth	729 bp
BBa_K2926049	Mating factor alpha from S. cerevisiae fused to mCherry	Johanna Opgenoorth	762 bp
BBa_K2926050	Fusion protein of Flo11 from yeast and Cherry	Johanna Opgenoorth	1287 bp
BBa_K2926051	Fusion protein of Opy2 from yeast and Cherry	Johanna Opgenoorth	837 bp
BBa_K2926052	Fusion protein of mCherry and M13 bacteriophage protein pVIII	Johanna Opgenoorth	942 bp
BBa_K2926053	Fluorescence reporter mCherry fused to pVIII from M13 bacteriophage with purification tag	Johanna Opgenoorth	729 bp
BBa_K2926054	Fusion protein of mating factor alpha from yeast, mCherry, and pVIII from M13 bacteriophage	Johanna Opgenoorth	994 bp
BBa_K2926055	Fusion protein of Flo11 from yeast, mCherry, and pVIII from M13 bacteriophage	Johanna Opgenoorth	1521 bp
BBa_K2926056	Fusion protein of Opy2 from yeast, mCherry, and pVIII from M13 bacteriophage	Johanna Opgenoorth	1071 bp
BBa_K2926057	Fusion protein of mating factor alpha, mCherry, and pVIII with His-tag	Johanna Opgenoorth	1014 bp
BBa_K2926058	Fusion protein of Flo11 (yeast), mCherry, and pVIII (M13 bacteriophage) with purification tag	Johanna Opgenoorth	1539 bp
BBa_K2926067	Fusion protein of Opy2 (yeast), mCherry and pVIII (bacteriophage M13) with purification tag	Johanna Opgenoorth	1089 bp
BBa_K2926068	Proline-glycine-peptide fused to the n-terminus of mCherry	Johanna Opgenoorth	750 bp
BBa_K2926041	CmR resistance protein variant A part 1 (Cm_A_split1_NpuN (pSB3T5))	Nefeli Chanoutsi	402 bp
BBa_K2926042	CmR resistance protein variant A part 2 (NpuC_Cm_A_split2 (pSB1K3))	Nefeli Chanoutsi	678 bp
BBa_K2926076	CmR resistance protein variant B part 1 (Cm_B_split1_NpuN (pSB3T5))	Nefeli Chanoutsi	537 bp
BBa_K2926077	CmR resistance protein variant B part 2 (NpuC_Cm_B_split2 (pSB1K3))	Nefeli Chanoutsi	543 bp
BBa_K2926078	Kan resistance protein part 1 (Kan_split1_N_Intein (pSiMPI))	Nefeli Chanoutsi	630 bp
BBa_K2926079	Kan resistance protein part 2 (C_Intein_Kan_split2 (pSiMPI))	Nefeli Chanoutsi	582 bp
BBa_K2926080	Amp resistance protein part 1 (Amp_split1_N_Intein (pSiMPI))	Nefeli Chanoutsi	588 bp
BBa_K2926081	Amp resistance protein part 2 (C_Intein_Amp_split2 (pSiMPI))	Nefeli Chanoutsi	669 bp
BBa_K2926089	Hyg B resistance protein part 1 (Hyg_B_split1_NpuN (pSB1K3))	Nefeli Chanoutsi	1029 bp
BBa_K2926090	Hyg B resistance protein part 2 (NpuC_Hyg_B_split2 (pSB3T5))	Nefeli Chanoutsi	414 bp

References

Shah, Neel H.; Muir, Tom W. (2011): Split Inteins: Nature's Protein Ligases. In: Israel Journal of Chemistry 51 (8-9). Link

Zettler, Joachim; Schütz, Vivien; Mootz, Henning D. (2009): The naturally split Npu DnaE intein exhibits an extraordinarily high rate in the protein trans-splicing reaction. In: FEBS Letters 583. Link

Jillette, Nathaniel; Du, Menghan; Cheng, Albert (2019): Split Selectable Markers. In: bioRxiv. Link

Iwai, Hideo; Züger, Sara; Jin, Jennifer; Tam, Pui-Hang (2006): Highly efficient protein trans-splicing by a naturally split DnaE intein from Nostoc punctiforme. In: FEBS Letters 580 (7). Link

Cheriyan, Manoj; Pedamallu, Chandra S.; Tori, Kazuo; Perler, Francine (2013): Faster Protein Splicing with the Nostoc punctiforme DnaE Intein Using Non-native Extein Residues. In: Journal of Biological Chemistry 288 (6). Link

Oeemig, Jesper S.; Aranko, A. Sesilja; Djupsjöbacka, Janica; Heinämäki, Kimmo; Iwai, Hido (2009): Solution structure of DnaE intein from Nostoc punctiforme: Structural basis for the design of a new split intein suitable for site‐specific chemical modification. In: FEBS Letters 583 (9). Link

Dassa, Bareket; Amitai, Gil; Caspi, Jonathan; Schueler-Furman, Ora; Pietrokovski, Shmuel (2007): Trans Protein Splicing of Cyanobacterial Split Inteins in Endogenous and Exogenous Combinations. In: Biochemistry 46 (1). Link

Wei, Xin-Yuan; Sakr, Samer; Li, Jian-Hong; Wang, Li; Chen, Wen-Li; Zhang, Cheng-Cai (2006): Expression of split dnaE genes and trans-splicing of DnaE intein in the developmental cyanobacterium Anabaena sp. PCC 7120. In: Research in Microbiology 157 (3). Link

Sali, A., & Webb, B. (1989): MODELLER (Version 9.22). Retrieved from https://salilab.org/modeller/

Ingebrigtsen, Sveinung G.; Didriksen, Alena; Johannessen, Mona; Skalko-Basnet, Natasa; Holsaeter, Ann Mari (2017): Old drug, new wrapping – A possible comeback for chloramphenicol? In: International Journal of Pharmaceutics 526 (1-2). Link

Shaw, W. V.; Leslie, A. G. W. (1991): Chloramphenicol Acetyltransferase. In: Annual Review of biophysics. Link

Shaw, William V. (1983): Chloramphenicol Acetyltransferase: Enzymology and Molecular Biology. In: Molecular Biology 14 (1). Link

Pongs, O. (1979): Chapter 3: Chloramphenicol". In Hahn, eFred E. (ed.). Mechanism of Action of Antibacterial Agents. In: Antibiotics Volume V Part 1. Link

Edwards, Keith H. (2009): Optometry: Science, Techniques and Clinical Management. In: Elsevier Health Sciences. Link

Volkmann, Gerrit; Iwai, Hideo (2010): Protein trans-splicing and its use in structural biology: opportunities and limitations. In: Molecular BioSystems 11 Link

Wu, Hong; Hu, Zhuma; Liu, Xiang-Qin (1998): Protein trans-splicing by a split intein encoded in a split DnaE gene of Synechocystis sp. PCC6803. In: Proceedings of the National Academy of Sciences of the United States of America 95 (16) Link

Team:Bielefeld-CeBiTec/Basic Part