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Revision as of 02:32, 19 September 2015

Description

Overview

"Years from now we will treat most infections with bacteria and not antibiotics."

Professor James Malone-Lee
Barlow Professor of Geriatric Medicine

A solution is urgently needed for the increasing number of infections caused by antibiotic-resistant bacteria. The engineering of bacterial cells to fight human pathogens is a promising alternative to administering traditional antibiotics. Our project involves the use of synthetic biology to engineer living therapeutics that have the potential to treat urinary tract infections (UTIs), which are a major public health concern in developed countries. This is largely due to growing antibiotic resistance.

The Problem

  • Antibiotics have negative side effects and their resistance is a growing problem
  • UTIs are the most common hospital-acquired infection and the bacteria that cause them are frequently resistant to antibiotics
  • Antibiotic resistance in UTIs is caused by biofilms
  • Biofilms are currently estimated to be responsible for over 65% of all hospital-acquired infections
  • Current UTI treatments are ineffective and fail to prevent recurrent infections

Problem with Antibiotics

Antibiotic use is associated with numerous negative side effects, allergies and reactions. The most common side effects of antibiotics all impact the digestive system and occur in around one in ten people. Around one person in fifteen has an allergic reaction to antibiotics, especially penicillin and cephalosphorins [10]. Half of the patients we spoke to during our project wanted us to find an alternative to using antibiotics, owing to the severity of the negative side effects that they experience.

Despite their negative side effects, antibiotics have been used so widely and for so long that the targeted microbes have adapted to become resistant, reducing the effectiveness of the drugs. A vicious cycle ensues in which ineffective antibiotic treatments leads to overprescription and overexposure, which amplifies the problem of antibiotic resistance. Global antibiotic consumption grew by 30% between 2000 and 2010. [27] Described by the Chief Medical Officer of England as “a threat equal to that of terrorism”, the growing resistance to antibiotics is hindering the effective prevention and treatment of an ever increasing range of infections.

antibiotic resistance poses a catastrophic threat. If we don't act now, any one of us could go into hospital in 20 years for minor surgery and die because of an ordinary infection that can't be treated by antibiotics.

Professor Dame Sally Davies
Chief Medical Officer
March 2013

The World Health Organisation’s antibiotic resistance Global Report on Surveillance, reports increasing worldwide incidences of antimicrobial resistance, in particular antibiotic resistance. This highlights very high rates of resistance in bacteria that cause common healthcare associated and community-acquired infections, such as UTIs [1]. UTIs account for over 7 million doctor visits per year. Catheter associated UTIs (CAUTIs) are the most commonly acquired infection in hospitals, and there is a high incidence of antibiotic resistance in the bacteria that cause UTIs globally [2].

Biofilms

Biofilms are aggregates of surface-associated microorganisms that are encased in a matrix of highly-hydrated extracellular polymeric substances, which include extracellular polysaccharides, extracellular DNA, as well as proteins [6]. Van Leeuwenhoek, using his simple microscopes in 1684, first observed microorganisms on tooth surfaces and can be credited with the discovery of microbial biofilms. "The number of these animalcules in the scurf of a man's teeth are so many that I believe they exceed the number of men in a kingdom." - Leeuwenhoek

We now know a great deal more about biofilms. Environmental changes are responsible for the transition from planktonic growth to biofilm [13] and cause changes in the expression of surface molecules, virulence factors, and metabolic status. This allows the bacteria to acquire properties that enable their survival in unfavourable conditions [14,15], such as in the presence of antibiotics. The low nutrient and oxygen levels at the bottom of the biofilm give rise to metabolically-inactive bacteria. As such, these persister cells are rendered unsusceptible to most traditional antibiotics, which rely on bacterial metabolism to exert cell-killing effect [7].

UTIs

Biofilms are currently estimated to be responsible for over 65% of nosocomial infections and 80% of all microbial infections [16]. Bacterial biofilms play an important role in UTIs. UTIs are caused by the pathogenic invasion of the urinary tract, which causes an inflammatory response of the urothelium. It is estimated that approximately 40% of women have had a UTI at some time in their lives [10]. UTIs may be caused by a variety of different organisms, most commonly bacteria. The most frequent cause of UTI in adult women is Escherichia coli, accounting for approximately 85% of community-acquired UTIs and 25-50% of hospital-acquired UTIs. Nosocomial infections may involve more aggressive organisms such as Pseudomonas aeruginosa and Enterobacter species.

The Solution

  • Break down bacterial biofilms to liberate the bacteria encased within and reduce the dose of antibiotics required
  • Breaking down bacterial biofilms and kill the bacteria encased within to provide an alternative to antibiotics

Overview

Our solution is focused on providing a treatment for UTIs because conventional antibiotics are unable to treat these and other biofilm-associated infections. Given the prevalence of such infections, there is a growing need for alternative therapeutic agents that can specifically degrade biofilms and kill the bacteria encased within. The use of synthetic biology to produce enzymes is the most effective way to achieve this specificity based on current technology. Our solution aims to investigate how bacterial biofilm disrupting proteins and antimicrobial proteins can be exported from E. coli and subsequently retain their antibiofilm/antimicrobial function. Using this secretion device we seek to create a system that offers persistent protection against biofilm formation.

We have designed a device that can exert antibiofilm and antimicrobial activity against E. coli and P. aeruginosa, the two leading causes of CAUTIs [4]. A nonpathogenic laboratory strain of E. coli is used as the expression host for the production of these enzymes as a proof-of-concept. The antibiofilm enzymes that we are using are Dispersin B and Micrococcal DNase, and the antimicrobial proteins that we are using are Art-175 and Microcin S.

Degrading the Biofilm

Prof. Malone-Lee stressed to us that sensitivity is a greater problem than complete antibiotic resistance. “Many more strains of bacteria are just insensitive to low doses of antibiotics, many can be overcome by high doses over long periods of time. Resistance is definitely not absolute.” Breaking down the biofilm increases the sensitivity of the bacteria embedded within it. Planktonic bacteria are metabolically active and are thus prone to antibiotics, meaning that lower doses are required.

DspB (BBa_K1659200) is an enzyme produced by Aggregatibacter actinomycetemcomitans, a species of bacteria found in the human oral cavity that grows almost exclusively in the form of biofilms. Structural analysis of Dispersin B showed that the enzyme only works specifically against the β-1,6-glycosidic linkages found in poly-N-acetylglucosamine, which is a polysaccharide structural element found in the biofilms of E. coli but not in P. aeruginosa. An additional enzyme would need to be used to target the polysaccharide component of P. aeruginosa biofilms.

Micrococcal DNase (BBa_K1659300) is an endo-exonuclease that non-specifically catalyzes the hydrolysis of single- and double-stranded DNA under basic conditions and in the presence of Ca2+ ions, and is known to be able to speed up DNA hydrolysis by up to 1016 times [17]. We are using DNase to break down the extracellular DNA component of biofilms.

Killing the Bacteria

Although antibiotic resistance is not absolute, it does pose a very big threat to the effective treatment of many infections. The insensitivity of bacteria to antibiotics can also be attributed to increasing antibiotic resistance. As described above, antibiotics also have many side effects that reduce patient quality of life and decrease the likelihood of completing a course of antibiotics. With all of this in mind, our solution does not only break down the biofilm, but also kills the bacteria embedded within so as to provide an alternative to antibiotics.

Art-175

Art-175 (BBa_K1659000) derive their name from “artificial endolysins”. Endolysins are bacteriophage-encoded peptidoglycan hydrolases that pass through the cytoplasmic membrane, degrading the peptidoglycan layer and inducing the lysis of the infected cell.

MccS

MccS (BBa_K1659100) is a type of microcin, a subclass of antibacterial proteins known as bacteriocins. Microcins are small, enterobacteria-produced bacteriocins that exert antibacterial activity against closely-related species, and MccS is produced by E. coli present in the probiotic drug Symbioflor 2 that has been shown to successfully treat gastrointestinal disorders.

For more information, please visit our Parts page.

Current clinically-relevant pathogens have not been seen to exhibit resistance against our antimicrobial proteins of choice. Art-175 has been experimentally shown to be not susceptible to resistance development, likely because it targets the structural element of the bacterial cell wall that is highly conserved across species and difficult to mutate [8]. The mechanism by which Microcin S exerts antimicrobial activity is still currently unknown, but no bacterial strains except for the original strain of probiotic E. coli which produces Microcin S has been shown to be resistant to it thus far [9].

Secreting the Proteins

The proven secretion of folded, functional proteins across both bacterial cell membranes is a challenge for present day microbiologists. Our solution requires that we can export DspB, DNase, Art-175 and MccS out of the expression host and into the local biofilm environment. To achieve this, signal sequences are fused to the enzymes to target them for export through natural E. coli secretion pathways. Using this mechanism we can direct our anti-biofilm and antimicrobial agents at a biofilm infected surface as they are being produced.

DsbA

DsbA is a oxidoreductase protein found predominantly in Gram-negative bacteria, which functions as a protein-folding factor [19, 20]. The 2-19 peptide sequence of DsbA is a signal sequence that can direct passenger proteins for co-translational export via the signal recognition particle pathway [21, 22]. It has recently been shown that the DsbA signal sequence is capable of mediating passenger protein secretion under a selection of different induction temperatures [23].

Parts: BBa_K1659002, BBa_K1659201,BBa_K1659301

YebF

YebF is a 13kDa protein of unknown function that is perhaps the only protein that has been conclusively documented to be secreted into the extracellular medium by a laboratory E. coli strain. At the N-terminus, YebF has a 2.2 kDa sec-leader sequence which mediates its translocation through the bacterial inner membrane via the Sec pathway, and is cleaved upon translocation into the periplasm to give the 10.8 kDa "mature" form [24]. Export from periplasm into the extracellular space takes places via the Omp pathway. YebF has been used successfully to mediate the secretion of recombinant proteins [25,26].

Part: BBa_K1659003

Fla

Flagellin are the constituent subunits of the helical filament substructure of bacterial flagella. In the flagellar-building process, flagellin are exported out of the cell sequentially by the flagellum-specific export apparatus. F. Vonderviszt et al. demonstrated through their work that the signal sequence responsible for allowing the flagellar export system to identify and export Salmonella flagellin is its 26-47 amino acid residue segment [18].

Part: BBa_K1659001

Results

Antimicrobials

Art-175

BBa_K1659000 Art175

BBa_K1659001 Fla-Art175

BBa_K1659002 DsbA-Art175

BBa_K1659003 YebF-Art175

Art-175 has been shown in the literature kill Gram-negative bacteria via permeabilization of outer membrane followed by lysis of peptidoglycan layer. Expression of Art-175 in literature done in E. coli as host, implying that upon production Art-175 is not able to bypass inner membrane to reach peptidoglycan and exert lytic activity. We have cloned our Art-175 constructs into E. coli as a proof-of-concept.

Induction of expression of Art-175 should not kill expression host, whereas the secretion-tagged Art-175 should be able to lyse peptidoglycan layer and kill expression host cell upon initial export through inner membrane (DsbA- and YebF-) or straight to the extracellular medium (Fla-).

Figure 1: Toxicity assay. Expression host cell cultures were grown in a 96-well plate at 30°C with 200 rpm shaking. MG1655 pBAD/HisB is E. coli MG1655 having a blank pBAD/HisB plasmid transformed into it, and serves as the negative control in the experiment.

Figure 2: Expression host cell cultures were grown in a 96-well plate at 37°C with 200 rpm shaking. MG1655 pBAD/HisB is E. coli MG1655 having a blank pBAD/HisB plasmid transformed into it, and serves as the negative control in the experiment.

Figures 1 and 2 show that the expression of Art-175 with no YebF secretion tag exerts no killing effect. The expression of YebF-Art175 shows a large drop in OD600. This data suggests that the YebF tag is directing Art-175 for secretion across at least the inner membrane to reach the peptidoglycan layer and exert lytic activity.

Figure 3: Toxicity assay. Expression host cell cultures were grown in a 96-well plate at 30°C with 200 rpm shaking. MG1655 pBAD/HisB is E. coli MG1655 having a blank pBAD/HisB plasmid transformed into it, and serves as the negative control in the experiment.

Figure 4. Toxicity assay. Expression host cell cultures were grown in a 96-well plate at 30°C with 200 rpm shaking. RP437 ∆FliC pBAD/HisB is E. coli RP437 ∆FliC having a blank pBAD/HisB plasmid transformed into it, and serves as the negative control in the experiment.

Figures 3 and 4 show that the expression of DsbA-Art175 and Fla-Art175 at 30°C exerts little cell killing effect. This suggests that the DsbA and Fla secretion tags do not work at 30°C.

Figure 5: Toxicity assay. Expression host cell cultures were grown in a 96-well plate at 27°C with 200 rpm shaking. MG1655 pBAD/HisB is E. coli MG1655 having a blank pBAD/HisB plasmid transformed into it, and serves as the negative control in the experiment.

Figure 6: Toxicity assay. Expression host cell cultures were grown in a 96-well plate at 27°C with 200 rpm shaking. RP437 ∆FliC pBAD/HisB is E. coli RP437 ∆FliC having a blank pBAD/HisB plasmid transformed into it, and serves as the negative control in the experiment.

Figures 5 and 6 show that expression of DsbA-Art175 and Fla-Art175 at 27°C exerts a cell killing effect. Combined with the figures 3 and 4 this data suggests the DsbA and Fla secretion tags function better at lower temperatures. For flagellar secretion, this data can be tentatively explained by high-temperature inhibition of flagellum synthesis.

Figure 7: P. putida incubated with supernatants in a 96-well plate at 27°C with 200 rpm shaking. The non-induced supernatant was prepared using the exact same steps as the induced supernatant, except Milli-Q water being added to the culture in place of L-ara.

Having shown that Fla-Art175 at 27°C kills host cells significantly, a culture was then expressed at a higher temperature (30°C) to sustain host cell growth while trying to maximize expression and secretion.

Figure 7 shows that the isolated supernatant of Fla-Art175 exerts effective cell killing activity against P. putida. This suggests that the Fla secretion tag is directing Art-175 for flagellar secretion across both cell membranes, and suggests that Art-175 is folded and functional in the extracellular environment.

Figure shows that YebF-Art175 causes host cell lysis even when induction is only started at mid-log. A lot of cell debris could be seen in the flasks.

As the construct is causing host cell lysis, we opted to upgrade the chassis to the more durable Yersinia enterocolitica. YebF-Art175 (pBAD/HisB) was transformed into Y. enterocolitica. IML421asd by postdoc Dr Diepold. Experimental steps involving live cultures of Yersinia all performed by Dr Diepold.

Figure 8: The non-induced supernatant was prepared using the exact same steps as the induced supernatant, except Milli-Q water being added to the culture in place of L-ara.

This data shows that the isolated supernatant of YebF-Art175 exerts effective cell killing activity against P. putida. This suggests that the YebF secretion tag is directing Art-175 for Sec mediated secretion across both cell membranes, and suggests that Art-175 is folded and functional in the extracellular environment able to kill the target cells but refrain from killing the host cells. We conclude that this part works as expected, with host cell lysis occuring to a large extent and the supernatant of gene expression subcultures being able to kill both planktonic and biofilm-encased (persister) P. putida.

MccS

BBa_K1659100 MccS

We showed that it is toxic to E. coli RP437 ∆FliC but not MG1655. This adds to the argument presented in the original literature that Microcin S’ antimicrobial capacity is narrow-spectrum and has some degree of strain specificity. More cells are killed at 30°C than 37°C, likely a combination of higher peptide efficiency and lower growth rate at that temperature.

Figure 9: Expression host cell cultures were grown in a 96-well plate at 30°C with 200 rpm shaking.

Figure 10: Expression host cell cultures were grown in a 96-well plate at 37°C with 200 rpm shaking.

From the data we can clearly draw 2 conclusions: 1. MccS is more toxic to RP437 than MG1655, to the extent where even low induction (0.002% arabinose) inhibits cell growth in RP437 almost entirely. 2. MccS appears more toxic at 30°C than 37°C.

Antibiofilms

DspB

BBa_K1659200 and BBa_K1659210: DspB

BBa_K1659201 and BBa_K1659211: DsbA-DspB

All DspB assaying done using BBa_K1659211

Having identified that secretion is successful, we also want to investigate if the concentration of enzyme in the supernatant is high enough to prevent planktonic wild-type cultures forming biofilms and to degrade pre-formed biofilms. We expect the expression hosts for these parts to form less biofilms when expression is induced.

Figure 10: SDS-PAGE of supernatant of MG1655 DsbA-DspB with expression, purified using divalent nickel column. Lane D is where the stained eluate was loaded (DsbA-DspB is a 45kDa protein); Ladder used was 2-Color SDS Marker

Figure 10 shows DsbA signal sequence successfully facilitates the export of DspB from MG1655.

Figure 11: Inhibition of host cell biofilm formation

Figure 11 shows that expression of DsbA-DspB inhibits host cell biofilm formation.

Figure 12: Co-culturing of Host Cell with normal biofilm-forming E. coli to inhibit normal E. coli from forming biofilms

Figure 12 shows that MG1655 DsbA-DspB[pBAD] inhibits the normal biofilm-forming E. coli from forming biofilms to a certain extent.

We conclude that this part works as we have conclusively proven: - Secretion of hexahistidine-tagged DsbA-DspB using nickel-affinity chromatography - That secreted DsbA-DspB inhibits biofilm formation of expression host - That secreted DsbA-DspB inhibits biofilm formation of normal biofilm-forming E. coli present in co-culture

DNase

BBa_K1659301

Figure 13: SDS-PAGE of nickel-affinity column chromatography purification of hexahistidine tagged DsbA-DNase. Lane C is where the stained eluate was loaded (DsbA-DNase is a 21kDa protein); Ladder used was 2-Color SDS Marker

Figure 13 shows that the DsbA signal sequence successfully facilitates the export of DNase from MG1655.

Figure 10 (above) shows that expression of DsbA-DNase inhibits host cell biofilm formation.

Improving the function of another team’s part: BBa_K729004

Figure 14: SDS-PAGE of E. coli MG1655 BBa_K729004 [pBAD], 0% ara supernatant (A) and E. coli MG1655 BBa_K729004 [pBAD], 0.2% ara supernatant (B)

Figure 14 shows the successful DsbA-directed secretion of DNase across both cell membranes.

A is the supernatant of uninduced E. coli MG1655 BBa_K729004 [pBAD], whilst B is the supernatant of 0.2% induced E. coli MG1655 BBa_K729004 [pBAD]. The band is approximately 21 kDa, corresponding to the size of DsbA-DNase.

Figure 15: Expression host MG1655 BBa_K729004 [pBAD] biofilm growth assay

Figure 15 shows the effect of inducing the expression of BBa_K729004 [pBAD] on the ability of the host to form biofilms. The control (MG1655, pBAD/HisB, 0.2% ara) and MG1655, BBa_K729004[pBAD], 0% ara are both able to grow biofilms, as shown by the intensity of the crystal violet staining. When BBa_K729004[pBAD] is expressed, the intensity of the crystal violet staining is reduced, showing a diminished ability to grow biofilm. This data suggests that the secretion of DNase is able to inhibit biofilm formation.

Conclusion

Future

Our modelling of diffusion out of the AlgiBeads showed that we would need too great a number of AlgiBeads for this to be realistic design approach. To overcome this problem we would need unrealistically efficient enzymes or unrealistically high protein synthesis rates. The other way around this is to create a system that provides a high local concentration of antibiofilm/antimicrobial agents at the point that is needed.

To develop our project beyond a proof-of-concept design, we would adopt a more suitable chassis, such as Lactococcus lactis. L. lactis has been widely used as a host for the production of proteins to target Gram-negative pathogenic bacteria. Using E. coli as our host was purely a starting point. We predict that moving to L. lactis to secrete our antibiofilm/antibacterial agents will overcome the problem of induced host cell lysis.

In addition to secreting antibiofilm/antimicrobial proteins, a comprehensive treatment for UTIs would be a bacteria engineered to also sense and move towards biofilms. We conducted extensive literature review on this in the early stages of the project but, due to the time restraints of a summer project, could not put our ideas into practice. With further work, we would incorporate both a sensing and chemotaxis mechanism into our design.

Our modelling of diffusion out of the biobeads showed that we would need too great a number of biobeads for this to be realistic design approach. To overcome this problem we would need unrealistically efficient enzymes or unrealistically high protein synthesis rates. The other way around this is to create a system that provides a high local concentration of antibiofilm/antimicrobial agents at the point that is needed. A high local concentration could be achieved by introducing free-living bacteria into the urinary tract. This idea is explored extensively in the Practices part of the project.

Nurses, doctors and professors all raised to us the issue of targeting the multiple bacterial and fungal species that are involved in UTIs, highlighting the fact that the problem extends further than E. coli and P. aeruginosa. Again, we have explored how we would approach this in the Practices page.

References

  1. Global Report on Surveillance of Antimicrobial Resistance: 2014. WHO.
  2. Johnson, J.R., 2004. Laboratory diagnosis of urinary tract infections in adult patients. Clinical infectious diseases : an official publication of the Infectious Diseases Society of America, 39(6), p.873; author reply 873–874.
  3. Zalewska-Piatek, B. et al., 2013. Biochemical characteristic of biofilm of uropathogenic Escherichia coli Dr+ strains. Microbiological Research, 168, pp.367–378.
  4. Sievert, D.M. et al., 2013. antibiotic-resistant pathogens associated with healthcare-associated infections: summary of data reported to the National Healthcare Safety Network at the Centers for Disease Control and Prevention, 2009-2010. Infection control and hospital epidemiology : the official journal of the Society of Hospital Epidemiologists of America, 34(1), pp.1–14. Available at: http://www.ncbi.nlm.nih.gov/pubmed/23221186.
  5. Fux, C. a. et al., 2005. Survival strategies of infectious biofilms. Trends in Microbiology, 13(1), pp.34–40.
  6. Flemming, H.-C. & Wingender, J., 2010. The biofilm matrix. Nature reviews. Microbiology, 8(9), pp.623–633. Available at: http://dx.doi.org/10.1038/nrmicro2415.
  7. Høiby, N. et al., 2010. Antibiotic resistance of bacterial biofilms. International Journal of antibiotic Agents, 35(4), pp.322–332.
  8. Briers, Y. et al., 2014. Art-175 is a highly efficient antibiotic against multidrug-resistant strains and persisters of Pseudomonas aeruginosa. antibiotic Agents and Chemotherapy, 58(7), pp.3774–3784.
  9. Gunzer, F., 2013. Bacterially-formed microcin S, a new antibiotic peptide, effective against pathogenic microorganisms, e.g. enterohaemorrhagic Escherichia coli (EHEC), European Patent EP2557163A1.
  10. Antibiotics - Side effects. Avaolable from: http://www.nhs.uk/Conditions/Antibiotics-penicillins/Pages/Side-effects.aspx [5/06/2015]
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  14. L. Zhang and T. Mah, “Involvement of a novel efflux system in biofilm-specific resistance to antibiotics,” Journal of Bacteriology, vol. 190, no. 13, pp. 4447–4452, 2008.
  15. J. Klebensberger, A. Birkenmaier, R. Geffers, S. Kjelleberg, and B. Philipp, “SiaA and SiaD are essential for inducing autoaggregation as a specific response to detergent stress in Pseudomonas aeruginosa,” Environmental Microbiology, vol. 11, no. 12, pp. 3073–3086, 2009
  16. U. Römling and C. Balsalobre, “Biofilm infections, their resilience to therapy and innovative treatment strategies,” Journal of Internal Medicine, vol. 272, no. 6, pp. 541–561, 2012
  17. Hale, S.P., Poole, L.B. & Gerlt, J. a, 1993. Mechanism of the reaction catalyzed by staphylococcal nuclease: identification of the rate-determining step. Biochemistry, 32(29), pp.7479–7487
  18. Vondervizst, F., Sajó, R., Dobó, J., & Závodszky, P. (2012). The Use of a Flagellar Export Signal for the Secretion of Recombinant Proteins in Salmonella. In: Recombinant Gene Expression - Reviews and Protocols, Methods in Molecular Biology, 824, 131-143.
  19. Guddat, L.W., Bardwell, J.C. & Martin, J.L., 1998. Crystal structures of reduced and oxidized DsbA: investigation of domain motion and thiolate stabilization. Structure (London, England : 1993), 6(6), pp.757–767.
  20. Heras, B. et al., 2009. DSB proteins and bacterial pathogenicity. Nature reviews. Microbiology, 7(3), pp.215–225.
  21. Schierle, C.F. et al., 2003. The DsbA signal sequence directs efficient, cotranslational export of passenger proteins to the Escherichia coli periplasm via the signal recognition particle pathway. Journal of Bacteriology, 185(19), pp.5706–5713.
  22. Steiner, D. et al., 2006. Signal sequences directing cotranslational translocation expand the range of proteins amenable to phage display. Nature biotechnology, 24(7), pp.823–831.
  23. Božić, N. et al., 2013. The DsbA signal peptide-mediated secretion of a highly efficient raw-starch-digesting, recombinant α-amylase from Bacillus licheniformis ATCC 9945a. Process Biochemistry, 48(3), pp.438–442.
  24. Zhang, G., Brokx, S. & Weiner, J.H., 2006. Extracellular accumulation of recombinant proteins fused to the carrier protein YebF in Escherichia coli. Nature biotechnology, 24(1), pp.100–104.
  25. Fisher, A.C. et al., 2011. Production of secretory and extracellular N-linked glycoproteins in Escherichia coli. Applied and Environmental Microbiology, 77(3), pp.871–881.
  26. Hwang, I.Y. et al., 2014. Reprogramming microbes to be pathogen-Seeking killers. ACS Synthetic Biology, 3(4), pp.228–237.
  27. Dramatic rise seen in antibiotic use. Available from: http://www.nature.com/news/dramatic-rise-seen-in-antibiotic-use-1.18383?WT.mc_id=TWT_NatureNews [17/09/2015]