NCBI Bookshelf. A service of the National Library of Medicine, National Institutes of Health.

Molecular Imaging and Contrast Agent Database (MICAD) [Internet]. Bethesda (MD): National Center for Biotechnology Information (US); 2004-2013.

Cover of Molecular Imaging and Contrast Agent Database (MICAD)

Molecular Imaging and Contrast Agent Database (MICAD) [Internet].

Show details

Luciferase-expressing Escherichia coli

pLux-expressing E. coli
, PhD
National Center for Biotechnology Information, NLM, NIH, Bethesda, MD 20894, vog.hin.mln.ibcn@dacim

Created: ; Last Update: February 5, 2008.

Chemical name:Luciferase-expressing Escherichia coli
Abbreviated name:pLux-expressing E. coli
Synonym:Light-emitting E. coli
Agent Category:Escherichia coli
Target:Non-specific tumor
Target Category:Bioluminescence
Method of detection:Optical imaging
Source of signal:Bioluminescence luciferase-expressing plasmid pLux
  • Checkbox In vitro
  • Checkbox Rodents
Click for the nucleotide sequence and the amino acid sequence of subunit A and subunit B of Photobacterium leiognathi luciferase.



A variety of therapies such as radiation, chemotherapy, recombinant biological drugs, surgical resection, or a combination of these regimens may be, and often are, used to treat neoplastic conditions. However, although promising to some extent, none of these treatments assures a successful outcome for the patient. It is also known that early detection and treatment of cancer greatly improves the odds of a favorable outcome. Therefore, medical researchers and professionals have devoted much effort to the development of different techniques and modalities such as positron emission tomography, single-photon emission computed tomography, magnetic resonance imaging, ultrasound, etc., for the detection and diagnosis of cancers (1-4).

Interestingly, the detection of different bacteria in tumors removed from patients has been documented, and some investigators have attempted to use the bacteria as a means to detect and target neoplastic tumors (5, 6). The bacteria appear to accumulate, survive, and thrive under the hypoxic and necrotic conditions found in the tumors, whereas they do not grow in normal tissues that have a rich supply of oxygen (7). The spores of an anaerobic strain of Clostridium novyi, created by removing the lethal toxin gene in the bacteria, germinated in the avascular regions of the tumor and subsequently destroyed the tumor cells (7). In addition, the ability of these bacteria to accumulate and grow in tumors has been exploited by using them as a signal source to image tumors (8). To achieve this, the bacteria were genetically engineered to express luciferase (Lux) or green fluorescence protein (GFP) genes and used to target and visualize tumors in mice (8). By engineering the luxCDABE gene cluster into the bacteria, a continuous light-emitting microbe was generated that did not need an external stimulus or substrate for the emission of light (8, 9). The emission of light is an energy-dependent process, the mechanism of which has been detailed elsewhere (10).

The use of Salmonella typhimurium for the treatment of cancers has been evaluated in clinical trials approved by the United States Food and Drug Administration (11, 12)

This chapter briefly describes the use of Escherichia coli transduced with a pLux plasmid that expresses the luciferase gene for the imaging of xenograft tumors in mice (13). After intravenous administration of the pLux-expressing E. coli, bioluminescence from the microbes could be detected exclusively in the tumor tissue 24 h later.



The E. coli strains MG1655 and HJ1020 and the plasmids used for this study were as described by Min et al. (13). The bacterial strains were generated by using the P1 transduction protocol as described by Silhavy et al. (14). The E. coli strain HJ1020 (asd::kan) was constructed from MG1655 as detailed elsewhere (15). The asd open reading frame in the plasmid pKD13 was replaced with kan by polymerase chain reaction (PCR) amplification with the use of appropriate primers. Subsequently, kan was removed to generate ∆asd as described by Datsenko and Wanner (15). The pLux plasmid was constructed by ligating a 9.5-kb XbaI fragment from pT7-3-Lux (a plasmid that contains the luxCDABE sequence obtained from Photobacterium leiognathi (16)) into the same site in the pUC19 plasmid so the lux operon could be driven by the lac promoter.

To construct the plasmid containing the dual Lux and asd cassette, the asd gene was PCR-amplified from E. coli MG1655, and the 1.1-kb fragment was cloned into the plasmid pGEM-T Easy that had been digested with EcoRI and ligated into the same site in pLux to generate the Asd+ pLux construct (13). The asd gene codes for aspartate β-semialdehyde dehydrogenase, and to survive the E. coli asd mutants have an obligate requirement for diaminopimelic acid, without which the cells are lysed. Therefore, bacterial cells containing the Asd+ pLux construct would be expected to survive longer compared to those harboring only the pLux plasmid.

In Vitro Studies: Testing in Cells and Tissues


No references are currently available.

Animal Studies



Tumors were visualized with the use of light-emitting E. coli in BALB/c and BALB/c athymic nu-/nu- mice bearing subcutaneous tumors generated with CT26, C6, SNU-C5 and B16-F10 cells in the thigh or right shoulder of each animal (13). To determine the optimal number of bacterial cells necessary for noninvasive imaging, different numbers of E. coli were injected subcutaneously into mice bearing tumors and the bioluminescence signals were acquired. The minimum number of E. coli required for imaging was determined to be 104, and the signal correlated well for up to 109 bacteria administered to the animals.

The biodistribution of E. coli that expresses the lux gene was determined in the mice bearing CT26 cell tumors (13). The distribution of E. coli carrying either the pLux or the Asd+ pLux in nude mice (n > 6) was investigated. The animals were infected by an intravenous injection of 108E. coli through the tail vein, and whole-body images were acquired. During the first few hours the signal was detected primarily in the liver and spleen of the animals, but by 24 h the signal was reduced in these organs and was detected exclusively in the tumor region of the animals. The investigators reported that the bioluminescence of the bacteria containing the pLux plasmid in the tumor peaked by day 4, and it decreased to undetectable levels after that time (probably because of a loss of the plasmid from the cells). However, tumors with E. coli containing the Asd+ pLux plasmid maintained the bioluminescence levels for up to 20 days after inoculation. The bacterial numbers in the normal tissue and the tumors were determined to investigate any correlation between the bacterial number and imaging. Presence of the bacteria in the tissue was confirmed by growing serial dilutions of tissue homogenates on agar plates containing ampicillin. Immediately after inoculation with the bacteria, the highest bacterial count was evident in the spleen, liver, and lungs of the animals (2.0 × 104/g tissue to 1.6 × 107/g tissue) with a low count in the tumor (9.5 × 102/g tissue), but within 24 h the number of bacteria in the tumor increased >1,000-fold, reaching ~108/g tissue by day 4 after inoculation. During the same period the number of bacteria in the spleen, liver, and lungs decreased, and the bacteria were undetectable in these organs at day 4. This indicated that the bacteria were cleared from the normal organs but accumulated and proliferated in the tumors.

Confocal microscopic examination of the tumor tissue revealed that the E. coli bacteria were present between the central necrotic part and the peripheral proliferative region of the lesion, but they were not detected in the peripheral proliferative region of the tumor (13). The investigators reported observing a higher number of tumor necrosis cases in the infected versus in the uninfected mice, and Min et al. suggested that the intravenously injected bacteria spread from the central necrotic region of the tumor to the peripheral regions.

The bioluminescent bacteria were observed to accumulate in a variety of tumors regardless of size, location (subcutaneous, intracecal, or shoulder, etc.), or immune status because the tumors could be detected both in the nude and the immunocompetent mice (13). In addition, the bacteria were observed to accumulate in the tumors whether administered through an intravenous or by an intraperitoneal inoculation.

In another study, the investigators established a metastatic tumor model in mice using the 4T1 murine breast cancer cells that metastasize specifically to the liver and the lungs (13, 17). As a control, a 4TO7 murine breast cancer cell line that does not metastasize was used to produce tumors. The cells were injected into the right fat pads of the BALB/c mice to establish an orthotopic breast cancer and metastasis (13). Four weeks after injection, E. coli Asd+ pLux were intravenously injected into the animals, and the bioluminescence signal was traced for three days. Immediately after inoculation, bacterial bioluminescence was detected in the liver for both tumor types. By two days after inoculation, the bioluminescence was also detected in the primary tumor sites (fat pads in the abdomen), but only the mice carrying the metastatic 4T1 tumor cells exhibited continuing bioluminescence in the liver, which indicates the presence of metastasis in the organ (13). The metastasis was confirmed by a histological examination, and such lesions were absent from the liver of control animals. From these observations the investigators concluded that bioluminescent E. coli is capable of imaging both primary and metastatic cancers (13).

Other Non-Primate Mammals


No references are currently available.

Non-Human Primates


No references are currently available.

Human Studies


No references are currently available.

Supplemental Information



Chong S. , Lee K.S. Spectrum of findings and usefulness of integrated PET/CT in patients with known or suspected neuroendocrine tumors of the lung. Cancer Imaging. 2007;7:195–201. [PMC free article: PMC2151326] [PubMed: 18055292]
Stillebroer A.B. , Oosterwijk E. , Oyen W.J. , Mulders P.F. , Boerman O.C. Radiolabeled antibodies in renal cell carcinoma. Cancer Imaging. 2007;7:179–88. [PMC free article: PMC2151324] [PubMed: 18055291]
Richards P.S. , Peacock T.E. The role of ultrasound in the detection of cervical lymph node metastases in clinically N0 squamous cell carcinoma of the head and neck. Cancer Imaging. 2007;7:167–78. [PMC free article: PMC2151323] [PubMed: 18055290]
Morana G. , Salviato E. , Guarise A. and , Contrast agents for hepatic MRI. Cancer Imaging, . 7 Spec No A: p. S24-7. 2007 [PMC free article: PMC2727962] [PubMed: 17921081]
Pawelek J.M. , Low K.B. , Bermudes D. Bacteria as tumour-targeting vectors. Lancet Oncol. 2003;4(9):548–56. [PubMed: 12965276]
Wei M.Q. , Mengesha A. , Good D. , Anne J. Bacterial targeted tumour therapy-dawn of a new era. Cancer Lett. 2008;259(1):16–27. [PubMed: 18063294]
Dang L.H. , Bettegowda C. , Huso D.L. , Kinzler K.W. , Vogelstein B. Combination bacteriolytic therapy for the treatment of experimental tumors. Proc Natl Acad Sci U S A. 2001;98(26):15155–60. [PMC free article: PMC64999] [PubMed: 11724950]
Yu Y.A. , Timiryasova T. , Zhang Q. , Beltz R. , Szalay A.A. Optical imaging: bacteria, viruses, and mammalian cells encoding light-emitting proteins reveal the locations of primary tumors and metastases in animals. Anal Bioanal Chem. 2003;377(6):964–72. [PubMed: 12879198]
Fernandez-Pinas F. , Wolk C.P. Expression of luxCD-E in Anabaena sp. can replace the use of exogenous aldehyde for in vivo localization of transcription by luxAB. Gene. 1994;150(1):169–74. [PubMed: 7959046]
Thorn R.M. , Nelson S.M. , Greenman J. Use of a bioluminescent Pseudomonas aeruginosa strain within an in vitro microbiological system, as a model of wound infection, to assess the antimicrobial efficacy of wound dressings by monitoring light production. Antimicrob Agents Chemother. 2007;51(9):3217–24. [PMC free article: PMC2043229] [PubMed: 17638701]
Toso J.F. , Gill V.J. , Hwu P. , Marincola F.M. , Restifo N.P. , Schwartzentruber D.J. , Sherry R.M. , Topalian S.L. , Yang J.C. , Stock F. , Freezer L.J. , Morton K.E. , Seipp C. , Haworth L. , Mavroukakis S. , White D. , MacDonald S. , Mao J. , Sznol M. , Rosenberg S.A. Phase I study of the intravenous administration of attenuated Salmonella typhimurium to patients with metastatic melanoma. J Clin Oncol. 2002;20(1):142–52. [PMC free article: PMC2064865] [PubMed: 11773163]
Nemunaitis J. , Cunningham C. , Senzer N. , Kuhn J. , Cramm J. , Litz C. , Cavagnolo R. , Cahill A. , Clairmont C. , Sznol M. Pilot trial of genetically modified, attenuated Salmonella expressing the E. coli cytosine deaminase gene in refractory cancer patients. Cancer Gene Ther. 2003;10(10):737–44. [PubMed: 14502226]
Min J.J. , Kim H.J. , Park J.H. , Moon S. , Jeong J.H. , Hong Y.J. , Cho K.O. , Nam J.H. , Kim N. , Park Y.K. , Bom H.S. , Rhee J.H. , Choy H.E. Noninvasive Real-time Imaging of Tumors and Metastases Using Tumor-targeting Light-emitting Escherichia coli. Mol Imaging Biol. 2008;10(1):54–61. [PubMed: 17994265]
Silhavy T.J. , Berman M.L. , Enquist L.W. and , Experiments with gene fusion. , Cold Spring Harbour Laboratory Press.: New York. 1984
Datsenko K.A. , Wanner B.L. One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products. Proc Natl Acad Sci U S A. 2000;97(12):6640–5. [PMC free article: PMC18686] [PubMed: 10829079]
Lee C.Y. , Szittner R.B. , Meighen E.A. The lux genes of the luminous bacterial symbiont, Photobacterium leiognathi, of the ponyfish. Nucleotide sequence, difference in gene organization, and high expression in mutant Escherichia coli. Eur J Biochem. 1991;201(1):161–7. [PubMed: 1915359]
Aslakson C.J. , Miller F.R. Selective events in the metastatic process defined by analysis of the sequential dissemination of subpopulations of a mouse mammary tumor. Cancer Res. 1992;52(6):1399–405. [PubMed: 1540948]


Search MICAD

Limit my Search:

Related information

  • PMC
    PubMed Central citations
  • PubMed
    Links to PubMed

Similar articles in PubMed

See reviews...See all...

Recent Activity

Your browsing activity is empty.

Activity recording is turned off.

Turn recording back on

See more...