Summary
Nowadays two plasmid systems are commonly used in the laboratory. In these Scenarios at least two antibiotics are necessary to enable stable upkeep of both plasmids in the cell. However, extensive use of antibiotics is known to promote the rise of antimicrobial resistance. This poses a growing threat, as a continued rise in resistance is predicted to cause 10 million deaths per year in 2050 (O’Neill, 2014). In addition usage of high levels of antibiotics is cost intensive and detrimental to industrial processes.
What if we had an alternative bypassing the need to use two antibiotics for retention of two plasmids in the same cell? To enable this, we split the antibiotic resistance gene for hygromycin and cloned each part into a different vector. After transforming cells with both vectors, active resistance protein is reconstituted through intein-mediated trans-splicing.
Hygromycin B (Fig. 1) 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).
Resistance
One of the enzymes that mediates resistance against hygromycin B (hygB) is called hygromycin-B 4-O-kinase and was found in Escherichia coli (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. 2). 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).
Inactivation of hygromycin B by ATP-dependent phosphorylation by hygromycin-B 4-O-kinases (Hph—Hygromycin-B 4-O-kinase, Uniprot)
Nostoc punctiforme DnaE Intein
sNMR solution structure of Nostoc punctiforme DnaE (PDB code: 2KEQ) (Oeemig et al. 2009)
An intervening protein element, short intein, is a part of a protein that excises itself from the precursor protein and fuses its flanking sequences, called exteins, with a peptide bond through protein trans-splicing (Anraku, Mizutani, & Satow, 2005). Thereby the amino acid sequence of the exteins directly besides the inteins crucially affects spicing efficiency, limiting the widespread use of many inteins in biology. The Nostoc punctiforme DnaE (Npu DnaE) intein is a subunit of the DNA polymerase III (DnaE) (Fig.3) from Nostoc punctiforme. In this organism the native protein splicing sites consists of the amino acids AEY on the N-extein and CFN on the C-extein (Cheriyan, Pedamallu, Tori, & Perler, 2013; Zettler, Schütz, & Mootz, 2009). In addition to those flanking sequences, Npu DnaE can also facilate protein splicing in a broad context of sequences. In some cases of synthetic extein sequences this results in even faster splice rates compared to the native ones (Cheriyan et al., 2013).
For more information about the difference between split and non-split inteins visit our part collection.
Design of Concept
Improved Part
For our project we improved the part BBa_K678021 encoding the Hygromycin-B 4-O-kinase. During the production of our Troygenics, we noticed that E. coli grows slower when simultaneously containing two plasmids. We reckon that the two antibiotics in media and the associated stress as well as the need for resistance protein production are major factors for E. coli’s slower growth rate and presumably for lower Troygenics product formation. Additionally the threat of multiresitant bacteria grows due to overuse of antibiotics in agriculture and medicine. This poses a grave problem and continued rise of multiresistant bacteria is assumed to cause 10 million annual deaths by 2050 (O’Neill, 2014). Therefore, we constructed plasmids the plasmids pSB1K3_Hyg1 and pSB3T5_Hyg2 each containing BBa_K2926089 and BBa_K2926090.
In detail our approach was to construct two plasmids that express the active hygromycin B resistance gene, only by the presence of both plasmids into one E. coli cell. To achieve this, we used intein-mediated trans-splicing of two parts of the hygromycin B resistance gene. To choose the optimal split point, we took the paper of Jillette, Du, & Cheng, 2018 into consideration. This restricted us to a specific site containing the amino acids relevant for the splicing process. To get sufficient splicing efficiency out of our Npu DnaE inteins we used information about the preferred flanking sequences from Cheriyan et al., 2013. Taken all this into consideration we chose the split point for the hygromycin resistance gene (HygR) between the amino acids 240 and 241.
Mechanism
The selection marker is split in two halfs. Each part of it is cloned into a different vector carrying distinct cassettes (Fig.4). 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 hygromycin-B 4-O-kinase. Due to this mechanism only cells containing both plasmids can grow on hygromycin medium.
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. In this case the process leads to active hygromycin-B 4-O-kinase, which conveys hygromycin B resistance.
Plasmid structure
As previously mentioned we determined the optimal split site for the hygromycin resistance gene (hygR) between the amino acids 240 and 241. To implement this split resistance we constructed two plasmids. The N-terminal part of the hygR gene and intein, respectively is located on the first plasmid. On the second plasmid the C-terminal part of hygR and intein were cloned (Fig. 5, 6). Upstream of the N-terminal Npu DnaE, the flanking amino acids in the extein from position -3 to -1 were W, L and A and downstream of the C-terminal intein from +1 to +3 C, M and E. These amino acids ensure a quicker splicing that the native intein-flanking site of Npu DnaE. To obtain roughly similar expression levels of both fusion proteins we cloned the longer and therefore presumably harder to expressed protein on the plasmid with the higher copy number because of the pUC ori. The coding sequence of the shorter fusion protein was cloned on the lower copy number plasmid, due the the p15A ori.
Fusion of the N-terminal part of HygR to the N-terminal Npu DnaE intein. The first 240 N-terminal amino acids HygR (HygR) are fused upstream of the N-terminal Npu DnaE intein. Between both domains on the positions -3, -2 and -1 of the splicing site the amino acids tryptophan, leucine and alanine (W L A) are located.
Fusion of the C-terminal Npu DnaE intein to the C-terminal part of the HygR. The last C-terminal aminoacids of HygR from position 241 are fused to the C-terminal Npu DnaE intein. Between both domains on the positions +1, +2 and +3 of the splicing site the amino acids cysteine, methionine and glutamic acids (C M E) are located.
Characterization
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 containing pSB1K3_Hyg1 (pSB1Ha3) or the C-terminal hygromycin resistance containing 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.
Single plasmid control
After the assembly of both plasmids, each containing pSB1K3_Hyg1 and pSB3T5_Hyg2, we transformed one plasmid into E. coli. Both strains were plated on LB plates containing 100 µg/mL of hygromycin B (Fig. 7). The chosen concentrations are well within the normal working concentrations for hygromycin B of 100 to 1000 µg/mL (Hygromycin B, Thermofisher). The E. coli strains containing only the N- or C-terminal part of hygR could not grow on hygromycin B plates. This shows, that the hygR termini alone are not sufficient to confer resistance against hygromycin B.
Growth control
Growth of E. coli containing both of our split hygR plasmids as well as suitable controls on media containing different amounts of hygromycin B. LB plates with different concentration of hygromycin B (40, 30, 60 80, 100, 200, 500, 1000 μg/mL, top left to botton right) were prepared. Multiple colonies of E. coli containing both of our split hygR plasmids, were plated on these plates (upper right third of each plate). In addition, E. coli DH5α (upper left third of each plate) and E. coli transformed with a plasmid containing full length hygR (lower third of each plate) were plated as negative and positive control respectively.
As we have established that only one plasmid and thus only one term of hygR is not sufficient to confer resistance to hygromycin B, in the next step we wanted to characterize our complete split resistance system. Therefore, we co-transformed E. coli cells with pSB1K3_Hyg1 and pSB3T5_Hyg2 and tested their efficiency of the reconstituted hygR by conducting growth experiments on plates with different concentrations of hygromycin B. The used concentration ranged from 30 to 1000 µg/mL (Fig. 8), while the normally used hygromycin B concentration is about 200 µg/mL (Hygromycin B, Thermofisher). The experiment showed, that our split hygR 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. coli DH5α did not grow on any hygromycin B concentration, while the positive control grew on all hygromycin B concentrations.
In conclusion, we were able to assemble the plasmids pSB1K3_Hyg1 and pSB3T5_Hyg2 each containing BBa_K2926089 and BBa_K2926090. We verified that only one plasmid with half of the resistance gene is not sufficient for the cell to grow on LB plates containing Hygromycin B. Finally, we showed that splitting the hygromycin resistance gene (HygR) into different vectors and fusing them to the Npu DnaE intein parts forms an active Hygromycin-B 4-O-kinase. Therefore, we established a two plasmid system based on only one antibiotic resistance. This enables a cheaper and more secure two plasmid based gene expression in prokaryotes for everyone to use.
References
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Cheriyan, M., Pedamallu, C. S., Tori, K., & Perler, F. (2013). Faster protein splicing with the Nostoc punctiforme DnaE intein using non-native extein residues. The Journal of Biological Chemistry, 288(9), 6202–6211. https://doi.org/10.1074/jbc.M112.433094
González, A., Jiménez, A., Vázquez, D., Davies, J. E., & Schindler, D. (1978). Studies on the mode of action of hygromycin B, an inhibitor of translocation in eukaryotes. Biochimica et Biophysica Acta (BBA) - Nucleic Acids and Protein Synthesis, 521(2), 459–469. https://doi.org/10.1016/0005-2787(78)90287-3
Gritz, L., & Davies, J. (1983). Plasmid-encoded hygromycin B resistance: The sequence of hygromycin B phosphotransferase gene and its expression in Escherichia coli and Saccharomyces cerevisiae. Gene, 25(2), 179–188. https://doi.org/10.1016/0378-1119(83)90223-8
Jillette, N., Du, M., & Cheng, A. (2018). Split Selectable Markers. BioRxiv, 452979. https://doi.org/10.1101/452979
Kaster, K. R., Burgett, S. G., Rao, R. N., & Ingolia, T. D. (1983). Analysis of a bacterial hygromycin B resistance gene by twraniptional and translational fusions and by DNA sequencing. Nucleic Acids Research, 17.
O’Neill, J. (2014). Antimicrobial Resistance: Tackling a crisis for the health and wealth of nations. Retrieved from https://amr-review.org/sites/default/files/AMR%20Review%20Paper%20-%20Tackling%20a%20crisis%20for%20the%20health%20and%20wealth%20of%20nations_1.pdf
Stogios, P. J., Shakya, T., Evdokimova, E., Savchenko, A., & Wright, G. D. (2011). Structure and Function of APH(4)-Ia, a Hygromycin B Resistance Enzyme. The Journal of Biological Chemistry, 286(3), 1966–1975. https://doi.org/10.1074/jbc.M110.194266
Zettler, J., Schütz, V., & Mootz, H. D. (2009). The naturally split Npu DnaE intein exhibits an extraordinarily high rate in the protein trans-splicing reaction. FEBS Letters, 583(5), 909–914. https://doi.org/10.1016/j.febslet.2009.02.003
