Summary
Would you love working with a two-plasmid system using only one antibiotic resistance? We enable you to do this by splitting antibiotic resistance genes with intein-mediated protein trans-splicing. By implementing this innovative approach, the likelihood of spreading resistances is decreased as one plasmid does not encode the full protein. Therefore, a single plasmid is insufficient to confer an antibiotic resistance to bacteria in the environment, functioning as a biosafety system. To ensure the universal applicability within the iGEM community we selected resistances against commonly used antibiotics: Chloramphenicol, Kanamycin, Ampicillin and Hygromycin B. The optimal split point was selected based on our model, taking into consideration the 3D-structure and the amino acids relevant for the splicing process. By constructing and characterizing a collection of two-plasmid systems we demonstrated the feasibility of our split-antibiotic system to maintain two plasmids in a single cell. This intein-based approach is transferable to other resistance markers.
Ampicillin (Fig. 1) is an antibiotic used to combat bacterial infections. Like other antibiotics, it can be administrated via mouth and injection into a muscle. It is classified under the category of bacteriolytic antibiotics and can penetrate gram-positive and gram-negative bacteria. It acts by inhibiting the transpeptidase, an enzyme responsible for cell wall formation in bacteria (Ampicillin Monograph for Professionals, 2018).
Ampicillin Resistance
Resistance against ampicillin in conferred by the enzyme beta lactamase. This enzyme is naturally produced by bacteria to defend themselves against beta-lactam antibiotics secreted by other microorganisms in the same ecological niche. The most common lactamases are the class A enzymes, such as the clinically significant TEM-1 lactamase (Waley, 1992). This enzyme class provides antibiotic resistance by hydrolysing the defining four-atom beta-lactam ring of the antibiotic, thus deactivating the molecule's antibacterial properties (Fig. 2).
Reaction schema of ampicillin hydrolysation by TEM-1 lactamase
Chloramphenicol
Chemical structure of chloramphenicol
A commonly used antibiotic for bacterial infections is chloramphenicol (Fig. 3)(Chloramphenicol Monograph for Professionals, 2019). It was isolated from Streptomyces venezuelae in 1947 and was the first artificially produced antibiotic (Pongs O.,1979). After it passes the cell membrane it has a bacteriolytic effect. It binds to specific residues of the 23S rRNA of the 50S ribosomal subunit, thus inhibiting the substrate binding in the ribosome (Chloramphenicol - Infectious Diseases, 2019) (Wolfe, A. D., 1965). Chloramphenicol is often one of the ingredients in eye ointments for the treatment of conjunctivitis (Edwards 2009). In some other cases it can be administered by mouth or vein injection (Rosenfield, Logan, & Edwards, 2009). Furthermore, it is used to treat hazardous bacterial infections like plaque, cholera, MRSA or typhoid fever (Ingebrigtsen 2017).
Ribbon structure of the chloramphenicol acetyltransferase with chloramphenicol bound to the catalytic center. (From PDB: 3CLA)(Leslie, A.G.W. 1990)
Chloramphenicol Resistance
The chloramphenicol acetyltransferase (CAT) is an enzyme originally identified in Escherichia coli ( E. coli) that mediates resistance to chloramphenicol (Fig. 4) (Shaw 1983). Its mode of action is the acetylation of chloramphenicol which abolishes chloramphenicols binding affinity to the 23S rRNA (Shaw 1991). Three quite distinct types of CAT enzymes, CATI, CATII and CATIII, are known. All types catalyze the same inactivation reaction (Fig. 5), but differ by varying degrees in their amino acid sequence and protein structure. The cat gene the iGEM community uses commonly, as part BBa_J31005 and in the standard backbone pSB1C3, codes for a type I CAT.
Chemical structures of chloramphenicol and its acetylation by chloramphenicol acetyltransferase.
Hygromycin B
Chemical structure of hygromycin B
Hygromycin B (Fig. 6) is an aminoglycoside that can kill prokaryotic and eukaryotic cells. It was developed in 1950 and used for animals as an anti-worming agent. This antibiotic is naturally produced by Streptomyces hygroscopicus and the gene responsible for synthesis of hygromycine B was discovered in 1980(Gritz & Davies, 1983; Kaster, Burgett, Rao, & Ingolia, 1983). Hygromycin B acts by inhibiting the enzymic translocation of peptidyl-tRNAs and thereby blocks the cytoplasmic protein synthesis (González, Jiménez, Vázquez, Davies, & Schindler, 1978).
Hygromycin B Resistance
One of the enzymes that mediates resistance against hygromycin B (hygB) is called hygromycin-B 4-O-kinase and was found in E. coli (Hph—Hygromycin-B 4-O-kinase, Uniprot). The enzyme inactivates hygromycin B by ATP-dependent phosphorylation and produces phosphor-hygromycin B and ADP (Fig. 7). This enzyme was also found in the organism Streptomyces fradiae, named APH(4)-Ia and was tested for it´s substrate spectrum (Stogios et. al., 2011). The test included numerous substrates like 4,6-disubstituted 2-deoxystreptamine-based, 4,5-disubstituted 2-deoxystreptamine-based and atypical aminoglycosides. Nevertheless the only antibiotic that was susceptible for phosphorylation by APH(4)-Ia was hygB. Such a narrow substrate spectrum is not common for APHs (Stogios et. al., 2011).
Kanamycin (Fig. 8) is a glycoside that was first isolated in 1957 from the bacterium Streptomyces kanamycericus (Sneader, 2005). It is used to treat bacterial infections and tuberculosis (Kantrex, 2019) and can be taken up by mouth, injection into a vein or into a muscle, but is ineffective against viral infections. The mode of actin for kanamycin is inhibition of cell growth by binding to the 30S subunit of the bacterial ribosome. This leads to incorrect alignment with the mRNA during translation and therefore to the assembly of a false amino acid sequence. That leads to the assembly of on functional peptide chains (Kantrex, 2019).
Kanamycin Resistance
Aminoglycoside-3'-phosphotransferase (APH(3')), also known as aminoglycoside kinase, is an enzyme that primarily catalyzes ATP-dependent phosphorylation of the 3'-hydroxyl group of 4,6-disubstituted aminoglycoside like kanamycin (Figure 4)(Gerard D. Wright, 1999). The specific variant of the aminoglycoside kinase we use the the gene aph(3’)-Ia. It provides resistance against kanamycin as well as neomycin and pradimicin (Shaw, 1993).
Intein-Mediated Protein Splicing
Inteins
Mechanism of intein-mediated protein splicing. The intein autocatalytically cleaves itself out of the polypeptide, connecting both adjacent exteins by a peptide bond .
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 (Fig. 9).
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 (Fig. 10).
Mechanism of split intein-mediated protein splicing. The N- and C-inteins rapidly associate together and form a functional intein, which mediates its own excision from the protein, thus joining the exteins by a peptide bond.
After the first split intein Synechocystis species DnaE (Ssp DnaE), a subunit of the DNA polymerase III (DnaE) from Synechocystis species 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 (Npu DnaE) which is highly homologous to Ssp DnaE was analyzed quite extensively. It has shown higher activities than Ssp DnaE 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).
Mechanism
Split-Resistance Genes
For the generation of split-resistance selection marker the coding-sequence is split in two parts. Each part is cloned into a different vector carrying distinct cassettes (Fig. 11). The markers N-terminal (MarN) and C-terminal (MarC) fragment is fused downstream and upstream respectively with the N-terminal (IntN) and C-terminal (IntC) fragment of the split Npu DnaE Intein. Each intein-extein fusion protein is expressed separately. In cells containing both plasmids the individual intein-extein fusion proteins find one another and connect the exteins by trans splicing, thus reconstituting the complete and active resistance protein. Due to this mechanism only cells containing both plasmids can grow on media containing hygromycin B.
Overview of two plasmid retention system using split selection markers. The selection marker is split in two parts and is fused upstream and downstream respectively to the N- and C-terminal fragment of the Npu DnaE intein. Each fusion is cloned in a different plasmid backbone with compatible origins of replication. On the protein level the split intein fragments find each other and connect their parts of the marker gene by protein trans-splicing, leading to a full-length and active resistance protein.
Fusion-Protein Structure
There are many important features, that must be taken into consideration while planning to construct a two-plasmid system with split antibiotics. Especially finding the right split point with the correct adjacent amino acids in the resistance genes coding sequence is crucial for success. In our endeavor to find optimal split points we talked to Mr. Albert Cheng, PhD, who suggested us to use data on the relative favorability of each amino acid at each relevant position neighboring the inteins (Tab. 1) (Cheriyan, 2013). Additionally, we assumed that it is important to make sure that the split point is not interrupting an important secondary structure of the protein. Correspondence with Francine Perler, PhD, reminded us that the exteins could also be hydrophobic as splitting a protein sometimes exposes the inner often hydrophobic amino acids to the polar surroundings. This could pose a problem if the exteins are too hydrophobic, leading to poor protein splicing efficiency. Instead of analyzing the DNA sequence of the resistance genes manually we decided to build a model, that will be able to predict the right split points.
As a positive reference to our collection of selected split points we contacted Prof. Dr. Barbara Di Ventura. She provided us with two plasmids encoding the N- and C-terminal part for a split TEM-1 lactamase and other two plasmids encoding the split coding sequence for the kanamycin resistance aph(3’)-Ia (Palanisamy et al., 2019).
Relative favorability of each amino acid i (rF(Ai)) 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(Ai) values the occurrence of each amino acid at each position was counted and 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 at the N-terminal extein
rF 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
Characterization
Single Plasmid Control
An important aspect, when constructing and working with a split-resistance gene system is to ensure, that single subparts of the split-resistance does not confer antibiotic resistance. Therefore, we transformed each plasmid containing a single part of a split-resistance pair separately into E. coli and plated the cells on plates with a low concentration of the corresponding antibiotic.
Normal working concentrations for chloramphenicol are between 25 and 170 µg/mL (ATCC_recommendations_antibiotic_concentrations, 2003). On a plate containing 5 µg/mL the E. coli strains containing single split-resistance parts (upper left: pSB3T5_CmA_1, upper right: pSB3T5_CmB_1, lower left: pSB1K3_CmA_2, lower right: pSB1K3_CmB_2 were plated (Fig. 12). None of the plated colonies grew, indicating that the single subparts of the split-resistance proteins are not sufficient to confer chloramphenicol resistance even in the used very low chloramphenicol concentration.
The same experiment was carried out for our hygromycin B resistance markers. Here the split-hygromycin resistance marker plasmids (pSB1K3_Hyg1 and (pSB3T5_Hyg2 were plated on media containing 100 µg/mL hygromycin B (Fig. 13) (normal working concentration for hygromycin B between 100 and 1000 µg/mL (Hygromycin B, Thermofisher). Here none of the plated colonies grew, again showing that one split-parts alone is not sufficient to confer antibiotic resistance.
Plating of E. coli strains containing only one split-CAT on media with 5 μg/mL chloramphenicol. E. coli strains with the plasmids pSB1K3_CmA_1 (pSB1Ca3) (upper left) and pSB1K3_CmA_2 (pSB3Cb5) (lower left) constituting the variant CmA of the split-cat as well as pSB3T5_CmB_1 (pSB1Cc3) (upper right) and pSB3Cd5 (lower right) constituting the variant CmB of the split-cat were plated on media containing 5 μg/mL chloramphenicol.
Growth of E. coli strains either with the N- or the C-terminal parts of the hygromycin resistance on media with hygromycin B. E. coli cells transformed either with the N-terminal hygromycin resistance pSB1K3_Hyg1 (pSB1Ha3) or the C-terminal hygromycin resistance pSB3T5_Hyg2 (pSB3Hb5) were plated on media with containing 100 µg/mL hygromycin B. The cell with the N-terminal part of hygromycin resistance were plated in the top row. The cell with the C-terminal part of hygromycin resistance were plated in the middle row.
Growth Control
After showing that one single plasmid with only one half of the split-resistance marker does not provide an antibiotic resistance, the next experiment was to analyze the growth efficiency of cells containing both plasmids of the two plasmid split-resistance system. Therefore, we poured plates with diverse concentrations of chloramphenicol and hygromycin B (Fig. 14, 15).
Regarding the chloramphenicol resistance we used concentrations between 0.1, 1, 7, 10, 20, 40, 60, 80, 100 μg/mL. Each plate was divided into three sections. On the upper right site, the colonies containing both plasmids of the split-resistance system were plated. This section was additionally divided into two segments, for the two different split point of chloramphenicol. On the left side the CmA version and on the right side the CmB version was plated. As negative control E. coliDH5α and as positive control E. coli containing pSB1C3 were plated in the upper left and lower segment respectively
The maximal chloramphenicol concentration on which E. coliDH5α grew was 0.1 μg/mL, while the E. coli with pSB1C3 grew on all chloramphenicol concentrations. The two split cat versions conferred resistance against chloramphenicol up to a concentration of 1 µg/mL and in other two repeats of the experiment up to a concentration of 5 µg/mL. On a purely visual basis the CmB version works a bit better than the CmA version. This can be explained by different extein sequences flanking the split-sites, thus leading to more efficient protein splicing and resistance protein formation.
Growth of E. coli containing both plasmids from the split-chloramphenicol resistance system as well as suitable controls on plates with different concentrations of chloramphenicol. LB plates with different amounts of chloramphenicol (0.1, 1, 7, 10, 20, 40, 60, 80, 100 µg/ml) were prepared and E. coli strains with both plasmids of the split-chloramphenicol resistance system were plated in the top right section. The section was further separated in CmA on top and CmB on the bottom half of the section. E. coliDH5α as a negative control was plated in the top left section and E. coli containing pSB1C3 as a positive control was plated in the bottom section.
Growth of E. coli containing both of our split hygromycin resistance plasmids as well as suitable controls on media containing different amounts of hygromycin B. LB plates with different concentration of hygromycin B (30, 40, 60 80, 100, 200, 500, 1000 μg/mL, top left to bottom right) were prepared. Multiple colonies of E. coli containing both of our split hygromycin resistance plasmids, pSB1K3_Hyg1 and pSB3T5_Hyg2, were plated on these plates (upper right section). In addition, E. coliDH5α (upper left section) and E. coli transformed with a plasmid containing full length hygromycin resistance (lower section) were plated as negative and positive control respectively.
For the hygromycin B split resistance test plates with a hygromycin B concentration ranging from 30 to 1000 μg/mL were prepared. On each plate the E. coli strain with both plasmids of the split hygromycin resistance, pSB1K3_Hyg1 and pSB3T5_Hyg2, as well as E. coliDH5α and E. coli containing pSB1C3 were plated.
The experiment showed, that our split hygromycin resistance enables E. coli growth up to a concentration of 500 μg/mL and with some difficulties even up to 1000 μg/mL. The negative control E. coliDH5α did not grow on any hygromycin B concentration, while the positive control grew on all hygromycin B concentrations.
For the Ampicillin split resistance test plates with an ampicillin concentration ranging from 5 to 300 μg/mL were prepared (Fig. 16). On each plate the E. coli strain with both plasmids of the split ampicillin resistance ( BBa_2926080 and BBa_2926081) as well as E. coliDH5α and E. coli containing pSB3K3 were plated.
The experiment showed, that split ampicillin resistance enables E. coli growth up to a concentration of 200 μg/mL. The negative control E. coliDH5α did not grow on any ampicillin concentration, while the positive control grew on all kanamycin concentrations.
Growth of E. coli containing both split ampicillin resistance plasmids as well as suitable controls on media containing different amounts of ampicillin. LB plates with different concentration of ampicillin (5, 30, 60, 80, 90, 100, 150, 200, 300 μg/mL, top left to bottom right) were prepared. Multiple colonies of E. coli containing both split ampicillin resistance plasmids, were plated on these plates (upper right section). In addition, E. coliDH5α (upper left section) and E. coli transformed with a plasmid containing full length ampicillin resistance (lower section) were plated as negative and positive control respectively.
Growth of E. coli containing both split kanamycin resistance plasmids as well as suitable controls on media containing different amounts of kanamycin. LB plates with different concentration of kanamycin (35, 40, 45, 50, 60, 70, 80, 90 μg/mL, top left to bottom right) were prepared. Multiple colonies of E. coli containing both split kanamycin resistance plasmids, were plated on these plates (upper right section). In addition, E. coliDH5α (upper left section) and E. coli transformed with a plasmid containing full length kanamycin resistance (lower section) were plated as negative and positive control respectively.
For the kanamycin split resistance test plates with a kanamycin concentration ranging from 35 to 90 μg/mL were prepared (Fig. 17). On each plate the E. coli strain with both plasmids of the split kanamycin resistance (pSiMPI_Kan1 and pSiMPI_Kan2) as well as E. coliDH5α and E. coli containing pSB3K3 were plated.
The experiment showed, that split kanamycin resistance enables E. coli growth up to a concentration of 90 μg/mL. The negative control E. coliDH5α did not grow on any kanamycin concentration, while the positive control grew on all kanamycin concentrations.
Growth Control in LB-Medium
In order to test the functionality of our split resistance genes, we conducted growth experiments with DH5α cells containing the two-plasmid system. The E. coli strains were cultivated in liquid LB media and all tested plasmids were cloned and transformed in DH5α cells. For every growth curve, there were five samples (Fig. 18). For the split kanamycin curve, the first sample was DH5α cells cultivated in LB-media without any antibiotics, the second DH5α culture in LB-kanamycin, the third DH5α + pSB1K3 in LB-kanamycin, the fourth DH5α + pSB1C3 + pSB3T5 in LB-chloramphenicol-tetracycline and the fifth DH5α cells with the split kanamycin system in LB-kanamycin.
DH5α cells with antibiotics did not grow in comparison to the DH5α sample without any antibiotics according to the expectations. The cells containing pSB1K3 grew the best while the split antibiotic system showed the second best growth curve. It was also observed that the cells containing two plasmids each with one antibiotic resistance did grow less efficient than the split kanamycin cells.
Growth experiment with E. coli containing different antibiotic resistances as well as the split antibiotic resistance system for experiments with the split kanamycin resistance system. DH5α cells with the split kanamycin system, DH5α + pSB1K3, DH5α + pSB1C3 + pSB3T5, DH5α in LB-medium with and without kanamycin (illustrated by squares, circles, triangles, reverse triangles and diamonds). The cells were grown at a temperature of 30 °C at 180 rpm.
The same procedure was conducted to test the functionality of the split ampicillin system. The cultures for these experiments were also cultivated in liquid LB media and all tested plasmids were cloned and transformed in DH5α cells (Fig. 19). The first sample was DH5α cells cultivated in LB-media without antibiotics, the second DH5α cells in LB-kanamycin, the third DH5α + pTXB1 in LB-ampicillin, the fourth DH5α + pSB1C3 + pSB3T5 in LB-chloramphenicol-tetracyclin, the fifth DH5α cells with the split ampicillin system in LB-ampicillin.
DH5α cells with antibiotics did not grow in comparison to DH5α cells without antibiotics, as shown before. The cells containing pSB1K3 grew as good as the cells with the split antibiotic system. DH5α cells with two antibiotic resistance genes grew worse than the others.
Growth experiment with E. coli containing different antibiotic resistances as well as the split antibiotic resistance system for experiments with the split ampicillin resistance system. DH5α cells with the split ampicillin system, DH5α + pTXB1, DH5α + pSB1C3 + pSB3T5, DH5α in LB-medium with and without kanamycin (illustrated by squares, circles, triangles, reverse triangles and diamonds). The cells were grown at a temperature of 30 °C at 180 rpm.
Modeling Split Chloramphenicol Resistance
We aimed to split the protein conveying resistance to chloramphenicol, chloramphenicol acetyltransferase (BBa_J3105). 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 (Fig. 20).
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).
As shown in growth experiments on plates, implementing split antibiotic resistances is feasible. Using the split points predicted with the model allowed us to successfully construct functional split antibiotic resistances.
Modeling Split Kanamycin Resistance
The amino acid sequence of the protein conveying kanamycin resistance, Aminoglycoside phosphotransferase APH(3')-Ia (Fig. 21) (Fong & Berghuis, 2002), was obtained from the iGEM-partsreg pSB1K3 and translated to a protein using ExPASy ( Expasy ).
The 3D-structure of Aminoglycoside Phosphotransferase APH(3')-Ia as obtained from PDB ( 4EJ7). This protein conveys resistance to kanamycin.
Using the python script, the ideal split point was identified as ETS-CSR at the amino acid position B(ETSCSR).
However, this split point is positioned rather close to the end of the polypeptide-chain. Moreover looking at the 3D-structure of the Aminoglycoside phosphotransferase APH(3')-Ia this position is marked to be a beta-strand, making it less likely that the splicing would result in the required protein structure.
The 3D-structure of the Aminoglycoside Phosphotransferase APH(3')-Ia. The peptide colored in magenta depict the split point identified as the most feasible one for Npu DnaE-intein-mediated splicing: ETS-CSR.
In addition to finding the optimal split point already within the sequence, we also considered adding one amino acid to broaden the possible combinations. We decided to implement a cysteine at the +1 position, since that is the most relevant position for the catayltic splicing mechanism, thus having a cysteine there greatly increases the likelihood of successful protein splicing.
The 3D-structure of the Aminoglycoside Phosphotransferase APH(3')-Ia. The peptides colored in magenta depict the position of two possible split points DDA-CWL (Fig. A) and GYK-CWA (Fig. B).
When identifying the ideal split points with a modification of the +1 amino acid to cysteine, our model found GYK-WA at position 25-29 and DDA-WL at position 91-95 to be the best split points.
The split point GYK-WA, with a B(GYKCWA) of 89.45, is still rather close to the end of the amino-acid chain (Fig. 23). Moreover, this position is located in the beginning of a beta-sheet.
The DDA-WL split point with a B(DDAWL) of 85.07. is still partly in beta-sheet. However, the splicing site is further away from the end of the polypeptide-chain than the GYK_WA position.
Since the B(Ai) values are in general higher for the intein-combinations with a newly introduced Cysteine, we decided to choose one of those.
As the best and second best predicted split points showed similar B value and were both located on the outskirts of beta-sheets we chose the GYK-WA position based on its position in the polypeptide chain. The more central split position in contrast to the DDA-WL position would increase the chance, that the splitting the protein would abolish the resistance activities of the individual subparts. Using only the last few amino acids as a split partner would pose the risk that the larger protein subpart would remain active and would thereby make our split resistance approach impossible. Therefore, we chose DDA-WL as our split point for Kanamycin.
Upon implementing this split point into the protein, the protein-parts combined with the intein were:
Part 1:
MSHIQRETSCSRPRLNSNLDADLYGYKWARDNVGQSGATIYRLYGKPDAPELFLKHGKGSVAND
VTDEMVRLNWLTEFMPLPTIKHFIRTPDDACLSYETEILTVEYGLLPIGKIVEKRIECTVYSVDNNG
NIYTQPVAQWHDRGEQEVFEYCLEDGSLIRATKDHKFMTVDGQMLPIDEIFERELDLMRVDNLP
NLEGHHHHHH
Part 2:
MIKIATRKYLGKQNVYDIGVERDHNFALKNGFIASNCWLLTTAIPGKTAFQVLEEYPDSGENIVDA LAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDASDFDDERNGWPVEQVWKEMHKLL PFSPDSVVTHGDFSLDNLDIFDEGKLIGCIDVGRVGIADRYQDLAILWNCLGEFSPSLQKRLFQKYG
IDNPDMNKLQFHLMLDEFFNLEGHHHHHH
Using homology modeling with MODELLER (Sali & Webb, 1989), the likely structure of each split part was determined. Next, the two protein parts were combined and adjacent to each other using Chimera, representing the protein structure prior to splicing.
The templates given were:
Part 1
Part 2
4QFQ, chain A (Npu DnaE)
4QFQ, chain B (Npu DnaE)
4EJ7, chain B (Aminoglycoside phosphotransferase APH(3')-Ia)
4EJ7, chain B (Aminoglycoside phosphotransferase APH(3')-Ia)
5OL6, chain B (inactivated Npu SICLOPPS intein with CAFHPQ extein)
5OL6, chain A (inactivated Npu SICLOPPS intein with CAFHPQ extein)
5OL7, chain A (inactivated Npu SICLOPPS intein with CFAHPQ extein)
4FEV, chain A (inactivated Npu SICLOPPS intein with CFAHPQ extein)
4KL5, chain B (Npu DnaE)
4FEU, chain A (Aminoglycoside phosphotransferase APH(3')-Ia)
Based on these templates, the default settings of MODELLER (Sali & Webb, 1989) were used to calculate a homology model from several templates.
The predicted 3D-structure of each subunit of the split Aminoglycoside phosphotransferase APH(3')-Ia prior to splicing. The split DDA-CWL was chosen based on our Modeling. The 3D-model was developed using the homology modeling software MODELLER (Sali & Webb, 1989).
split C-terminal intein, AmpR spit 2 and the insert TcR
References
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