Construct of DNA glucose sensor yeast plasmid for early detection of diabetes

Abstract

This investigation was aimed at assembling different genetic building blocks to produce a focused DNA sensor to detect proteins related to glucose in blood, in order to diagnose early diabetes using synthetic biology and conventional molecular biology. A glucose DNA sensor was constructed in Saccharomyces cerevisiae using genomic and synthesized sequences and were tested in both, In vivo using human blood plasma sampled, and In vitro using cultural media. The results were based on fluorescence intensity of the DNA sensor and compared with clinical methods. The DNA sensor was highly sensitive when mixed with plasma samples from different patients (i.e., diabetic, pre-diabetic, and normal) showing high fluorescence, and was able to detect a wide concentration range of glucose equivalent to clinical glycaemia values. Expression of proteins pertaining to glucose metabolism production was determined by 2D-DIGE gel electrophoresis-maldi analysis. The glucose sensor produced results less than a minute after being mixed with a drop of a human blood sample. Our results highlight the advantages of using constructed DNA sensors to detect glucose in blood for early diagnosis of diabetes.The yeast DNA glucose sensor was assembled successfully with standardized genetic parts and was able to detect low levels equivalent to clinical glycaemia (<140 mg/ dL), as well as a higher equivalent level of glycaemia (>200 mg/dL). Thus, our sensor can be used for early diagnosis of diabetes or pre-diabetic conditions, thereby allowing for earlier clinical intervention. The direct correlation between levels of glucose and the intensity of fluorescence of the DNA sensor shows the advantage of using this technology in order to identify specific types of diabetic patients. Sometimes, it is difficult to establish accurate correlations with different methods.human blood [7]. These types of sensors are versatile analytical tools that offer advantages over classical analytical methods due to their inherent specificity, selectivity, and simplicity [5]. Most biological and cellular sensors use membrane and cellular proteins that recognize and take up biomarkers from a medium [6]. However, DNA has also been used in molecular biosensors [8,9].Biomarkers for detecting and diagnosing diseases include physical symptoms, mutated DNA and RNA, secreted proteins, processes such as cell death or proliferation, and serum concentrations of small molecules such as cholesterol or, in the case of diabetes, glucose [6].Currently, the most common methods to diagnose diabetes use enzymatic reactions to determine glucose levels [10,11]. These methods measure glucose in whole blood because erythrocytes contain high concentrations of glucose [12]. The methods include the use of glucose oxidase, hexokinase, and glucose dehydrogenase [13][14][15]. The products of these reactions with blood sugar can be determined using colorimetric and spectrophotometric assays or by measuring the electric current produced in the enzyme reactions; the latter is the approach used ...

DNA-based glucose sensor and photonicity for early detection of diabetes using saliva

“…Clones with 100% DNA sequence accuracy were selected for further processing. The assembly of the construct DNA glucose sensor was also confirmed by PCR analysis, which showed full assembly of the different genetic parts [7].…”

“…Procedure: This procedure was carried-out by combining standard experimental labora-tory methods of molecular biology and synthetic biology [7] with proprietary methods from Clonetex Systems, Inc. (Austin, TX). Thus, PCR amplification was per-formed as follows: Each gene was PCR amplified using gene-specific overlap primers and assembled sequences were subcloned into a HingIII-and XbaI-digested pYES2 vector.…”

“…The fluorescence results were then divided in three groups according to the intensity of the fluorescence in relation to the clinical glycaemia concentration as described above [7] with the groups based on the means from ranges of fluorescence intensity. Samples were collected from 23 patients.…”

“…Assembly of the denoted DNA glucose sensor was effectively achieved, following standard molecular biology and synthetic biology procedures [7]. Thus, the different genetic parts of the sensor, which include Gal1 promoter and CYC1 terminator, along with the other genes SNF3, OGT, OGlcNA, and EGFP were fully integrated and subcloned into PYES2 vector (Figure 1).…”

“…Although these digital devices produce quick results, the range of sensitivity can still be enhanced [5,6]. Therefore, molecular glucose sensors based on DNA have been previously developed by the main author of the present paper [7] in order to enhance sensitivity for detection of glucose based on fluorescence emission following interaction of a DNA-based glucose sensor with saliva. Furthermore, the sensitivity of the DNA glucose sensor is enhanced by the additional inclusion of signaling and/or transport specific glycoproteins or proteins modified with glucose genes [8,9].…”