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  • Recombinant lactobacilli are being developed to serve as a bio-safely organism, in the hope to express heterologous protein in oral therapeutic applications.
  • In our project, caries nemesis "HOPE", we use lactobacillus casei as our final host of recombinant plasmid, which contains the promoters and the desired genes like YebF and Orf19. Orf19 and YebF are signal peptide and lysing protein which respectively derive from M102phage and E.coli MG1655. Also, low oxygen and acid environment were set as the initial condition to turn on our recombinant plasmid. Therefore, we gain an assess to do conditioned express of killing and anti-biofilm gene in L. casei.
  • However, the isolation of plasmid DNA from G+ lactobacilli is complicated by resilience of the peptidoglican layer.
  • For this reason, it is a relative feasible and reliable way that we use E. coli as our testing bacteria of “HOPE” but eventually, the functional promoters and genes of the shuttle vector will be transformed into probiotic L. casei., which is safe to use as a food-grade protein-secreting system.

Get started.

How to do it?

Expected results~


Before we get started:

The purpose of our study attempts to construct an E.coli-Lactobacillus shuttle vector plasmid with an expression cassette consisting of a strong promoter, a signal sequence, and a heterologous model protein gene. We put the plasmid inE. coli, our testing bacteria to conform the feasibility of our study and finally the plasmid will be inserted into L. casei in "HOPE".

Generally, lactic acid bacteria (LAB) are regarded as safe and useful commensal bacteria, which are known as probiotics and starters for food fermentation. In recent years there has been an upsurge of interest in the genetic manipulation of lactic acid bacteria (LAB). Many species are now transformable by electroporation and have thus become amenable to recombinant DNA technology.

For instance, Lactococcus lactis secreting biologically active interleukin 10 (IL-10) was established for the treatment of inflammatory bowel diseases in a murine model [1]. The successful study thereafter progressed to a clinical trial in humans [2]. For prevention of the transmission of human immunodeficiency virus type 1 (HIV-1), recombinant L. lactis and Lactobacillus plantarum that secrete microbiocidal cyanovirin-N were constructed and were capable of neutralizing the infectivity of HIV-1 in vitro[3]. These studies suggested that protein-secreting systems in LAB could be useful and offer a promising strategy for medical applications in the future.

Many studies on Lactobacillus casei subsp. have been carried out using the plasmid-free strain BL 23, ATCC 393 (pLZ15-)[4]. BL 23 and its relatives have been used as a host for genetic modification and applied for live delivery agents. For example, the single chain variable fragment of an antibody that binds to a major adhesion molecule of Streptococcus mutants was expressed on the cell surface and showed protective effects against colonization with the pathogen [5]. Thus, it is clear that the development of a highly efficient protein-secreting system in this strain would be important and beneficial.

Moreover, L. casei might be a better live delivery agent because the optimal growth temperature of L. casei is the same as the body temperature of mammals. Thus, BL 23 is no doubt a suitable bacteria host for our food-grade protein secreting system in “HOPE”.

So how did we do it?

Bacterial Strains and Growth Conditions

L. casei BL23 and recombinant strains were grown in Mann-Rogosa-Sharp (MRS) medium at 37℃.

Erythromycin (5 μg/ml) was added to MRS for the selection of recombinant L. casei strains. For pH control, 50 mM carbonate buffer was supplemented into the medium at certain ratios (NaHCO3:Na2CO3). The commonly used cloning host of E. coli DH5α(TaKaRa, Shiga, Japan) was grown in LB with or without 100 μg/ml ampicillin. L casei, BL 23 were maintained in a 10% glycerol stock collection at -80 °C and were subcultured in MRS medium (Oxoid) when required.

Bacterial strains used in this study:

Strain code  | Strain                       | Source
BL 23        | L. casei ATCC 393 (pLZ15-)   | [Y.C Tsai] NYMU, Taiwan

Construction of Plasmids and Transformation of L. casei

The sequences of all PCR primers used in this study are listed in the following Table 2. PCR products and plasmids were digested with restriction endonucleases followed by ligation and transformation to E.coli DH5α. In the cloning of E.coli DH5α we used a high copy number plasmid named E. coli, pUC19 After harvesting L. casei plasmid by digesting pUC19, we use a E-coli-Lactobacillus shuttle vector, pLP402, to perform our desired features. And the transfer process was carried out by electrophoresis.

PCR primers used in this study:

Primers   Sequences                                                         Restriction sites
IGM289     cccaagcttagatctgattacaaaggctttaagcagg                              HindIII, BglII
IGM290     gggctcgaggcccggttgttcgcggccgcttttgttaagaattttatttcataacattagcgg       XhoI, NotI
IGM291     cccgtcgacgcccggttgttcgcggccgcttcggttatactattcttgcttgata               SalI, NotI
IGM292     ggggaattcctgcagggatccaaacttgattgcataatctttcttcc                      BamHI, PstI, EcoRI
IGM350     acatattttatgtttggagggtattggatg
IGM351     catccaataccctccaaacataaaatatgt
IGM468     ccccggatccgagcaggggccagtacagccg                                      BamHI
IGM478     ccccctcgagttagcttttcattttgatcatcatgta                                 XhoI
IGM479     gcgaaatccaagcaaaggcgagcaggggccagtacagccg
IGM480     cggctgtactggcccctgctcgcctttgcttggatttcgc
IGM482     cggctgtactggcccctgctcggatccgagtttgtgtccgcctttgcttggatttcgc           BamHI

Expected results~


  1. Steidler, L., W. Hans, L. Schotte, S. Neirynck, F. Obermeier, W. Falk, W. Fiers, and E. Remaut. 2000. Treatment of murine colitis by Lactococcus lactis secreting interleukin-10. Science 289: 1352-1355.
  2. Braat, H., P. Rottiers, D. W. Hommes, N. Huyghebaert, E. Remaut, J. P. Remon, et al. 2006. A phase I trial with transgenic bacteria expressing interleukin-10 in Crohn’s disease. Clin. Gastroenterol. Hepatol. 4: 754-759.
  3. Pusch, O., D. Boden, S. Hannify, F. Lee, L. D. Tucker, M. R. Boyd, J. M. Wells, and B. Ramratnam. 2005. Bioengineering lactic acid bacteria to secrete the HIV-1 virucide cyanovirin. J. Acquir. Immune Defic. Syndr. 40: 512-220.
  4. Acedo-Félix, E. and G. Pérez-Martínez. 2003. Significant differences between Lactobacillus casei subsp. casei ATCC 393T and a commonly used plasmid-cured derivative revealed by a polyphasic study. Int. J. Syst. Evol. Microbiol. 53: 67-75.
  5. Krüger, C., Y. Hu, Q. Pan, H. Marcotte, A. Hultberg, D. Delwar, et al. 2002. In situ delivery of passive immunity by lactobacilli producing single-chain antibodies. Nat. Biotechnol. 20: 702-706.
  6. Kajikawa, Akinobu1, Eiko Ichikawa2, and Shizunobu Igimi2 2010. Development of a Highly Efficient Protein-Secreting System in Recombinant Lactobacillus casei. J. Microbiol. Biotechnol. (2010), 20(2), 375–382
  7. Zhengjun Chen & Jinzhong Lin & Chengjie Ma & Shumiao Zhao & Qunxin She & Yunxiang Liang Characterization of pMC11, a plasmid with dual origins of replication isolated from Lactobacillus casei MCJ and construction of shuttle vectors with each replicon. Appl Microbiol Biotechnol (2014) 98:5977–5989
  8. Evelia Acedo-Fe´ lix3 and Gaspar Pe´ rez-Martı´nez. Significant differences between Lactobacillus casei subsp. casei ATCC 393T and a commonly used plasmid-cured derivative revealed by a polyphasic study. International Journal of Systematic and Evolutionary Microbiology (2003), 53, 67–75