Progress Report RTD-Project GMOBILITY
Title: Safety evaluation of horizontal gene transfer from genetically modified organisms to the microflora of the food chain and human gut
Project: GMOBILITY (QLK1-CT-1999-00527)
Period: February - September 2000
Co-ordinator: Dr. Jos van der Vossen, TNO, The Netherlands
Risk evaluation of horizontal gene transfer (HGT) of marker genes to the human microflora and food bacteria.
To execute the project five work packages were defined. These are presented below including a brief description of the progress.
WP 1. Selection, construction and lab bench testing of donor sequences and recipient strains for horizontal gene transfer.
- Several donor systems were constructed:
A genetic construct containing the GFP (green fluorescent protein) gene was made in which GFP fluorescence is repressed. First experiments indicate the ability to discriminate GFP fluorescence from bacterial strains isolated from faeces of test mice carrying the GFP non repressed constructs in FACS (cell-sorting) analysis.
- Erythromycin resistance gene and luciferase (lux) reporter gene were selected as foreign genes to be introduced in Lactococcus lactis derivatives of IL1403. These two genes (Erm/Lux) were introduced i) into the chromosome of L. lactis under the control of a lactococcal promoter, ii) a non-conjugative plasmid derived from pAMbeta, and conjugative transposon Tn916. The construction of a conjugative plasmid with both genes is underway.
The Tn916 construct was used in transfer experiments. In in vitro conjugation assays any transfer of Tn916 derivative was observed from L. lactis to E. faecalis (10 E-9).
- Two genetically modified plants were selected as donor for the project: Genetically modified potato "Apriori" of AVEBE (the Netherlands) has become available and there is a request for GM Maize of Novartis (Swiss).
The bacterial species Acinetobacter sp., Escherichia coli and Bacillus subtilis have been chosen to perform transformation experiments and marker rescue experiments. For marker rescue an incomplete npt11 gene was selected. This gene is present in several GM-plants.
Initial studies for detection of green fluorescent bacteria, the FACS cell sorting system appeared to be promising.
WP 2. Horizontal gene transfer in food systems.
- For the investigation of gene transfer in food, strains of the species Acinetobacter sp., Escherichia coli and Bacillus subtilis have been chosen to perform transformation experiments and/or marker rescue experiments. For marker rescue experiments, we used three vectors that carry a partially deleted nptII gene. At present, we extend the number of the available vectors by subcloning the partially deleted nptII region into additional vectors that will allow broadening the host range for the marker rescue experiments. The first of these experiments with Acinetobacter sp. have been conducted successfully with either plasmid DNA or PCR fragments of the nptII gene as donor DNA. Experiments with plant DNA will be performed when genetically modified potatoes carrying the nptII gene become available.
- To facilitate the investigation of the persistence of plant DNA in different food matrices and under varying food-processing conditions, two PCR systems have been established. For genetically modified maize, the system permits to detect DNA fragments of the cry1A(b) gene of genetically modified maize with lengths between 211bp and 1913 bp. A similar PCR system for the EPSP (5- enolpyruvylshikimate-3-phosphate) synthase gene of genetically modified soy bean has been established, detecting fragments ranging between 172bp and 1719bp. This enables us to monitor the degradation kinetics of the plant DNA with regard to different environmental factors prevailing in food matrices.
- To monitor the kinetics of DNA degradation by the digestive process in TNOs rumen model a PCR test was developed. The target for the PCR is the npt111 gene with surrounding sequences. Fragments that can be detected with the PCR detection system is ranging from 113 bp to 2970 bp.
WP 3. Horizontal gene transfer in vitro systems.
- A start was made with the digestion studies on genetically modified potatoes in in vitro gastrointestinal tract model after ingestion of different food matrices. To monitor the kinetics of DNA degradation by the digestive process the PCR test is and will be implemented using the npt111 gene with surrounding sequences as target (described under WP2, point 3). If required, in view of the rapid DNA degradation process a nested PCR protocol is available. Such a protocol could enhance the detection level.
WP 4. Horizontal gene transfer in vivo.
- Transfer assays are now underway in mono-associated mice to test transfer of the Tn916 (Emr/ Lux)transposon in the digestive tract of mice.
- To study the stability and integrity of naked DNA in HFA rats different DNA isolation methods were tested for their suitability on rat fee, intestinal samples and faeces. Different methods were compared for the isolation of DNA from rat feed, intestinal samples and faeces. Three different DNA isolation methods (CTAB, Wizard Kit A, and Wizard genomic DNA purification kit) were tested on rat feed to obtain maximum yield and highest purity. The CTAB DNA isolation method proved to be best suitable for the rat feed. The presence of maize DNA in sterile rat feed was demonstrated by the invertase PCR. Fragments up to 400 bp were detected. The presence of GMO maize could not be shown. For the isolation of DNA from intestinal and faecal samples three methods were tested (Boom, CTAB, and a DNA isolation method for sediment samples). The best results were obtained with the Boom method. Neither fragments from the invertase gene nor from the cry1A(b) gene could be demonstrated in the intestinal and faecal samples.
To study the stability and integrity of naked DNA in HFA rats and food samples several PCR tests were developed. PCR tests were developed for the invertase and Cry1A(b) of Maize. For the semi-quantitative tests competitor DNA molecules were constructed and cloned in the vector pGEM-T for both the invertase and Cry1A(b) gene. Tested threshold levels were 0.1 %, 1%, 10% and 100%. A quantitative real-time PCR was developed for Bt-176 maize using the LightCycler of Roche. The used primers were derived from the invertase and the Cry1A(b) gene. Amplification of the targets was monitored by measuring the increase in fluorescence of the double stranded DNA binding dye SYBR Green I. The specificity of the amplified products was assessed by performing melting curve analysis. By this procedure it was demonstrated that the feed normally used for rats in the animal facilities at KUN contains 10 % of maize DNA. To test the stability and integrity of GMO maize DNA primers were designed to generate fragments of approximately 100 bp, 200 bp, 400 bp, and 800 bp from the maize invertase gene and cry1A(b) gene. The different fragments were cloned in the pGEM-T vector. The vector with the 800 bp cry1A(b) fragment was added to rat feed samples. After different time intervals DNA was isolated from the feed and the stability and integrity of the DNA was studied with the fragment-multiplex PCR. Integrity of maize DNA in various food samples will be tested. Several maize containing food products have been collected for this purpose. Both the invertase quantitative and fragment-multiplex PCR will be used.
Transfer assays are now underway in mono-associated mice to test transfer from GM bacteria.
To study the stability and integrity of naked DNA in HFA rats different DNA isolation methods were tested for their suitability on rat feed, intestinal samples and faeces. Maize DNA was shown present in sterile rat feed. GMO maize was not observed.
To study the stability and integrity of naked DNA in HFA rats and food samples several PCR tests were developed. PCR tests were developed for the invertase and Cry1A(b) of Maize.
Pilot feeding experiments were conducted in which rats were fed with non GM potatoes. Normal rat feed is now tested for the presence of npt11.
WP 5. Quantitative risk assessment and evaluation of model systems.
- There are no results available.