Design Problem 2 You wish to use microbial cells to produce large amount of some vitamin.

For this purpose you are asked to select a strong promoter. Which technique will you follow for the purpose? Expectations: a) Suggest only one technique to identify the promoter. b) Suggest one technique to isolate the promoter. c) Suggest some technique to detect whether actually production has increased or not. d) The solution should be such that it can be easily implemented. e) It is desirable to select a promoter which is not inhibited during any phase of growth

CONTENTS

INTRODUCTION
y y

Selection of Vitamin-Producing Microbes Identifying DNA Sequence/Gene for Vitamin Production

IDENTIFYING STRONG PROMOTER

y y y

Expression Vectors with Strong Promoters Isolation and Construction of the Promoter Insertion of Gene to Constructed Vector Analysis Fluorescent-Associated Cell Sorting (FACS)

DETECTION OF VITAMINS
y

SPR Biosensors

SOLUTION INTRODUCTION DNA replicates and then is transcribed to mRNA expressing particular gene. A promoter is the DNA sequence that initially binds the RNA polymerase (together with initiation factors in many cases). Once formed, the promoter-polymerase complex undergoes structural changes required for transcription initiation to proceed. In this problem, we would look for gene coding for enzyme that will act upon precursor for vitamin molecule and start the chain of reaction in a metabolic pathway converting one molecule into another eventually synthesising the vitamin. And then we would find suitable promoter sequence which would help DNA to transcribe to express that particular gene segment within that DNA sequence. Selection of Vitamin-Producing Microbes Bacteroides vulgatus and Escherichia coli are the microbial cells that can be used to produce Vitamin D, Vitamin B12, and Vitamin K etc. They can be isolated from intestinal tissues of humans or from any other external source. Species from the following genera are also known to synthesize vitamins: Acetobacterium, Aerobacter, Agrobacterium, Alcaligenes, Azotobacter, Bacillus, Clostridium, Corynebacterium, Flavobacterium, Micromonospora, Mycobacterium, Nocardia, Propionibacterium, Protaminobacter, Proteus, Pseudomonas, Rhizobium, Salmonella, Serratia, Streptomyces, Streptococcus and Xanthomonas. Streptomyces griseus is the most common commercial source of vitamin B12 production. But [Link] cells can be used for optimum production. Identifying DNA Sequence/Gene for Vitamin Production If we are particular about production of specific vitamin in excess amount, we must know the gene coding for precursor molecule of that particular vitamin or the enzyme that converts precursor to intermediate products carrying on the synthesis of vitamin. And since we know most of the vitamins and their function and role in growth, we can identify the gene sequence coding for enzyme converting precursor to vitamin molecule with the help of bioinformatics tools. We know all enzymes are proteins except lysozymes. So, first we need to identify genes transcribed and then translated to protein forming enzymes that catalyzes the reaction converting precursor molecule into vitamin. Considering we are interested in production of vitamin B 12 from [Link] cells; it contains genes hemB, hemC and hemD, encoding respectively the enzymes porphobilinogen synthase, hydroxymethylbilane synthase and uroporphyrinogen III synthase. These enzymes converts 5-aminolaevulinic acid (ALA) preparatively in high yield into uroporphyrinogen III. The Pseudomonas denitrificans genes cobA and cobI, encoding respectively the enzymes S-adenosyl-L-methionine:uroporphyrinogen III methyltransferase (SUMT) and Sadenosyl-L-methionine:precorrin-2 methyltransferase (SP2MT), can be expressed in E. Coli for few folds greater production of vitaminB 12.

Fig: biosynthesis of vitamin B12 (cobalmin)

[1-ALA; 2-PBG; 3-HMB; 4-uroporphyrinogen III; 5-precorrin-2; 6-precorrin-3A; 7-vitamin B12]

When SUMT is combined with the coupled-enzyme system that produces uroporphyrinogen III, precorrin-2 is synthesized from ALA, and when SP2MT is also added, the product from the coupling of five enzymes is precorrin-3A. Both of these products are precursors of vitamin B12, and they can be used directly for biosynthetic experiments.

IDENTIFYING STRONG PROMOTER Promoter is a DNA sequence responsible for initiation of transcription of a DNA segment into mRNA resulting in gene expression. Many of the precursor molecules of vitamin are glycol-proteins or lipids or steroids or mixture of any two groups. For example: cholesterol is a precursor for vitamin D. Identifying suitable strong promoter will involve comparison of vector constructed without promoters to that with vector constructed having genes to be expressed along with

promoter sequence with the help of Fluorescence-Associated Cell Sorting (FACS) technique using Green Fluorescent Protein (GFP).

 Expression Vectors with Strong Promoters

The main function of an expression vector is to yield the product of a gene- usually, more the product, the better it is. Therefore, expression vectors are ordinarily equipped with very strong promoters; the rationale is that the more mRNA that is produced, the more protein product will be made. One such strong promoter is the trp (tryptophan operon) promoter. It forms the basis for several expression vectors, including ptrpL1. It has a trp promoter/operator region, followed by a ribosome binding site, and can be used directly as an expression vector by inserting a foreign gene into the ClaI site. Alternatively, the trp control region can be made portable by cutting it out with ClaI and HindIII and inserting it in front of a gene to be expressed in another vector. This promoter is not inhibited during any phase of growth but the gene regulation by this promoter is inhibited only in the presence of tryptophan. To switch on the system, small molecules called inducers trigger the production of proteins by binding to the repressor protein and changing its conformation. This change alters the operator-repressor interaction, so that repressor can no longer remain attached to the operator.

 Isolation and Construction of the Promoter

A 0.5-kilobase-pair DNA fragment carrying the intact trp promoter is extracted by HinfI digestion from E. coli plasmid DNA, ptrp ED 5-1. It is cloned into the unique EcoRI site of pBR322, using EcoRI linker. The upstream EcoRI site was cleaved by the enzyme and filled in with T4 DNA polymerase. Then, the remaining downstream EcoRI site can be cleaved by EcoRI, followed by BAL-31 digestion to eliminate a sequence between the trpE-coding region and its Shine-Dalgarno (SD) sequence. A chemically synthesized Cla I linker will be inserted just downstream of the SD sequence. The resulting plasmid will contain sequence of the relevant region. It has the trp promoter, the SD sequence for trpL (attenuator), along with another SD sequence of trpE in front of the Cla I linker.

 Insertion of Gene to Constructed Vector

cobA and cobI genes can be isolated from genomic library once its sequence has been identified using bioinformatics tools. Length of gene cobA is around 842 base pairs and that of cobI is 737 base pairs. So these two genes combined can be cloned in plasmid vector. pBR322 is the vector used along with suitable strong promoter i.e. trp promoter. Its capability of strong promoter will be detected with the help of FACS using GFP.

Now, isolated cobA and cobI genes will be ligated to vector downstream to promoter sequence under optimum conditions. And the recombinant vector will be transferred to [Link] cells (host cells) cultured in the optimum growth medium.

 Analysis
y y y

Genomic library of pBR322 containing cobA and cobI genes can be created without a trp promoter sequence Next genomic library of recombinant pBR322 created containing the promoter sequence. Tagging the sequence with Green Fluorescent Protein (GFP): GFP had been widely used in labelling the DNA sequence, where expression of GFP can be used as a marker for a particular characteristic. GFP can also be expressed in different structures enabling morphological distinction. Here, the gene for the production of GFP is spliced into the genome of the organism in the region of the DNA that codes for the target proteins and that is controlled by the same regulatory sequence; that is, the gene's regulatory sequence now controls the production of GFP, in addition to the tagged protein(s). In cells where the gene is expressed, and the tagged proteins are produced, GFP is produced at the same time. Thus, only those cells in which the tagged gene is expressed, or the target proteins are produced, will fluoresce when observed under fluorescence microscopy. Fluorescence would be detected and the cells would be sorted out depending on their fluorescence with the help of fluorescence-associated cell sorting technique using flow cytometer.

Fluorescence-Associated Cell Sorting The process begins by placing the cells into a flask and forcing the cells to enter a small nozzle one at a time. The cells travel down the nozzle which is vibrated at an optimal frequency to produce drops at fixed distance from the nozzle. As the cells flow down the stream of liquid, they are scanned by a laser. If we want to separate a subpopulation of cells, we can do that by tagging those of interest with an antibody linked to a fluorescent dye. The antibody is bound to a protein that is uniquely expressed in the cells you want to separate. The GFP which we talked about earlier wont require any fluorescent dye to be linked to it but the working principle is same. The laser light excites the dye which emits a color of light that is detected by the photomultiplier tube, or light det ector. By collecting the information from the light (scatter and fluorescence) a computer can determine which cells are to be separated and collected.

Fig: Schematic Diagram of FACS

Fluorescence would be compared with GFP expression in vector containing trp promoter with those who do not contain it. The signals generated would be analyzed and the greater the signal generated (fluorescence) of GFP, the greater the gene is expressed under trp promoter or the promoter of gene itself. This will tell us how strongly the gene is expressed under the influence of trp promoter Vector pBR322 containing trp promoter will generate high mean fluorescence and thus our strong promoter would be identified.

DETECTION OF VITAMINS Vitamins can be detected and quantified by optical biosensor which exploits the phenomenon of surface plasmon resonance (SPR) to detect and measure bio-molecular interactions at an interface on a sensor chip. These are named SPR (Surface Plasmon Resonance) biosensor. The sensor monitors changes in the resonance angle which shifts when biomolecules bind to the chip surface and alter the refractive index of the surface layer. The biosensor methods are inhibition assays and detect vitamin binding proteins when they bind to the vitamin immobilised onto a sensor chip surface. The competition for binding with free vitamin molecules in the sample is measured as a change in response by the sensor.

SPR Biosensors SPR biosensors visualize the progress of biomolecular binding through time by defining the change in mass concentration that occurs on a sensor chip surface during the binding and dissociation process. The chips are composed of a glass surface coated with a thin layer of gold that provides the physical conditions necessary for the SPR reaction. Light is shone on the reverse side of this chip, propagating an electron charge density wave phenomenon that arises at the surface of the metallic film. This takes the form of an evanescent wave that extends beyond the sensor surface and detects mass changes on the surface. Light does not actually enter the sample at any time; thus, the quenching or absorbance problems characteristic of spectrophotometric and fluorescent techniques are eliminated. Flow conditions are controlled by using a liquid-handling system comprising precision pumps and an integrated fluidic cartridge that forms the flow cells on the chip surface and directs the flow of liquid over the surface.

Fig: SPR-based biosensors. The sensor chip provides a gold-coated surface through which minute changes in biomolecule concentration, due to molecular binding, are detected in real time. These changes lead to changes in the reflected light that are captured by the optical detection unit and converted to a resonance signal.

y y y y

The SPR biosensor monitors interactions of a specific binding protein with the vitamin immobilized on a CM5/gold sensor chip. The prepared samples are mixed with a fixed concentration of the vitamin binding protein by the auto-sampler and injected over the chip surface. The vitamin present in the sample binds to the protein and subsequently inhibits it from binding to the surface of the sensor chip. The higher the concentration of the vitamin is in the sample, the higher the level of inhibition and hence the lower the response of the biosensor. A regeneration step prepares the chip surface for the next sample. Quantification is performed by multi-level calibration with the vitamin standards.

Figure: SPR sensorgram. As molecules bind to and dissociate from the sensor chip surface, the resulting changes in the resonance signal create a sensorgram. The shape and amplitude of the measurements can then be used to determine overall concentration and the kinetics of the interaction

In this way, we can detect vitamin binding proteins for specific proteins. In this way we can detect increase in concentration of specific vitamin by doing biosensor assay with that particular vitamin only. We can perform this detection at the time when we first isolated the cells having recombinant DNA and then can perform it after interval of 30-45 minutes or so and determine the varying concentration of vitamin in the sample which should be increasing.