Genetic Engineering: Protein Synthesis

• The text is about gene technology

Making Proteins Using Genetic Engineering

• State different ways to produce a desired gene
• Explain importance of reverse transcriptase in producing genes for genetic engineering
• Explain the use of restriction enzymes to obtain desired genes
• Explain how to artificially make genes
• Explain plasmid properties making them suitable for gene cloning
• Explain why the location of promoters is close to genes needing transcription
• Explain why promoters need transfer with desired genes for modified cell production in eukaryotic hosts

Process of Making a Protein Using Genetic Engineering

• There are multiple steps involved
• Step 1 involves synthesizing or obtaining the desired gene
• Step 2 involves inserting a DNA fragment (gene) into a vector (carrier) using ligase enzyme, resulting in recombinant DNA
• Vectors include plasmids, viruses, and liposomes, and rely on sticky ends for insertion
• Step 3 transformation involves transferring the gene into a suitable host cell by calcium ions, cooling, heat shock and a promoter.
• Bacteria is transformed by taking a recombinant DNA
• Step 4 Identification of successfully transformed host cells is with gene markers
• Step 5 Involves large-scale production of the host cell population

Synthesizing/Obtaining the Desired Gene

• Use reverse transcriptase, extracting from bacteria, or synthesizing artificially
• Reverse transcriptase uses RNA as a template to produce DNA

Reverse Transcriptase Details

• Reverse transcriptase produces cDNA using mRNA as a template
• A cell selected readily produces the protein extracted
• These cells have large quantities of the required mRNA
• The enzyme makes a single DNA strand, cDNA, using mRNA template with a base sequence complementary to mRNA
• DNA polymerase makes another DNA strand, making the required double-stranded DNA
• Extracting specific mRNA that is present in many copies is easier
• Host cells such as bacteria might not have the metabolic capability of processing RNA transcripts with introns
• Mature mRNA produced does not have non-coding portions (introns)

Restriction Enzyme Use

• Restriction enzymes cut a gene from a chromosome
• Some bacteria produce restriction endonucleases, which cut up viral DNA, as a defense
• Restriction endonucleases each cut a DNA double strand at a specific base sequence called the recognition sequence
• Restriction enzymes can cut straight across the sugar-phosphate backbone, leaving blunt ends
• Restriction enzymes can cut DNA in a staggered fashion, leaving an uneven cut exposing unpaired bases (sticky ends)
• Palindromic sequences occur where two sequences are opposite of one another, leaving sticky ends

Synthesizing DNA Artificially

• It begins from nucleotides, analyzing the gene product (amino acid sequence), then synthesis the gene
• The genetic code is universal
• It is usually how vaccines are created

Insertion of DNA Fragments into Plasmids

• Plasmids are removed from bacterial cells and cut using a restriction endonuclease to create complementary sticky ends
• The same restriction enzyme should be used to cut both the plasmid and the desired gene
• If using cDNA (from reverse transcriptase) or synthesized DNA, sticky ends are added on
• To link DNA the sugar-phosphate backbone of the DNA molecule is linked to the plasmid used with DNA ligase
• The product is recombinant DNA

Plasmid Properties and Gene Cloning Suitability

• Plasmids are small pieces of double-stranded DNA (small circles of DNA) that can be inserted into a cell without contributing much additional DNA to the host cell
• They replicate independently in the host cell allowing cloning
• High copy numbers result in many copies of cloned genes
• Plasmids are easy to extract from bacteria and easy to be taken by bacterial cells
• Plasmids have a number of recognition sites allowing them to be cut by restriction/endonuclease
• DNA/gene can be inserted
• They possess gene markers like antibiotic resistance, which allows them to be readily recognized
• It is how transformed bacteria (bacteria that took up the recombinant DNA) are identified
• Plasmids are circular, reducing host cell degradation but are stable
• Plasmids act as vectors and may carry a promoter, so a gene can be expressed/transcribed

Transformation Process

• Transformation refers to gene transfer into a host cell, which can be achieved through various methods
• Bacteria are treated with a high concentration of calcium ions to affect cell wall and cell membrane permeability
• The bacteria are cooled for 30min and given a heat shock at 42°C for 30 seconds
• The process increases the chance of plasmids passing through the cell surface membrane
• Only about 1% takes up the plasmid with desired gene

Promoter Location Importance

• Close proximity allows initiating new gene transcription that allows RNA polymerase and transcription factors to bind
• It allow the RNA polymerase to recognize which of the two DNA strands will be the template (strand to be transcribed)
• RNA polymerase binds to allow transcription of the gene of interest, with transcription factors binding to initiate
• It also helps determine gene expression levels

Promoter Transfer Justification in Modified Cells (Eukaryotes)

• A gene must be inserted near an existing promoter
• If not, it could disrupt the expression of other genes
• Promoters need to be transferred with the desired gene when producing modified cells
• It can include non-coding DNA (introns) or or the gene might share a promoter with host gene not switched on

Why Use a Promoter?

• For initiating gene transcription, switching on the gene, or causing gene expression
• Allows RNA polymerase and transcription factors (TF) to bind
• Allows the RNA polymerase to recognize which of the two DNA strands will be the template strand for transcription
• Binds RNA polymerase for transcribing the gene of interest
• Determines gene expression level at a sufficient amount at the correct time
• Other genes must be inserted near existing promoters
• Genes can be inserted in any chromosome or included within introns
• A gene might share a promoter with a host gene, which does not switch on in the host cell

Role of Different Gene Markers

• Plasmids possess gene markers, often antibiotic resistance genes, that enable identifying if a cell has been transformed

Limitations of Antibiotic Resistance Genes

• There is risk of spreading resistance to other bacteria
• Antibiotics become ineffective

Blue-White Screening

• Genes produce protein/enzyme, and can easily be identified.
• Plasmids contain enzymes beta-glucuronidase (GUS) which originates from E.Coli
• GUS produce a colorless substance, what is easily stained

Fluorescent Proteins (GFP) Advantages

• It is easy to see a gene codes for a fluorescent protein
• It uses a protein from jelly fish make a green fluorescent protein
• The protein fluoresces under UV light
• Shining UV light allow identification of recombinant DNA (with plasmid).
• GFP's have no harmful effect on cells, so it is easier to identify cells
• The marker is inserted downstream next to a promotor region, so the gene maker and interest is transcribed together
• The protein production wont affect its function

Gene Coding for GFP

• GFP proteins are markers inserted into plasmids, downstream from a promoter to enable transcription
• It easily identified without harming the host cell
• By applying ultraviolet light visible green color fluorescence can be viewed

Gene Cloning and Production

• DNA polymerase in bacteria is the first that copies the copy plasmid, where replicas are independently produced with copies
• Replication by binary fission creates recombinant of each product and contains copies
• Then a gene translate into the required product, example insulin

Genetically Modified Bacteria Identification

• Bacteria are cultivated on agar plates containing ampicillin
• Only the plasmids with the antibiotic-resistant gene makes bacteria will resistant A,B
• Excluding them is the excluding of bacteria which are take nothing C
• With recombinant DNA, tetracyline is activated

Bacterial Identification

• Bacteria containing genes of resistance are tetracycline
• No activity has taken in a cell to grow with tetracycline

Modified Detection of Genetically Resistant Bacteria

• Vectors are removed from bacteria, and are the used for the restriction enzymes / or the PCR
• DNA link the sugar-phosphate in the Recombinant DNA
• Bacterial plasmid is transformed

Benefits of Genetically Modified Proteins

• Extracting data for insulin in pancreatic Beta cells
• mRNA is produced and obtains retroviruses
• Copy cDNA is produced through polymearse to assmble
• They sticky with the gene mix with plamsid and stick to
• Proteins can be linked through recombinant

Insulin Production Advantages

• The identical genetic code makes the production of the human identical.
• Response is more rapidly made
• Pure genetic code prevents potential from bacteria risk
• The increased production has no impact on ethical production

Factor VII for blood clotting anti hemophiliac

• Old methods uses the donated blood for the clotting which carried infection
• Nowadays the genetic has been modified by injection
• Factor are unlimited supply

Disadvantages of Producing Factor VIII

• The large supplies are extracted from blood transfusions
• The extracted factors can be damaged with the viral donation
• The gene is extracted so there is no rejection of infection
• Quick source can over come an ethical injection

##Electroporesis 3: Part

• This involves techniques like PCR and Electrophoresis

General Technique

• General tool to make copies of DNA( amplify)
• It is similar to semiconservative replication, it provides the visibility from the number of starting material
• Electroporesis is a rapid efficiency as its produces different copies

Primers

• Provide the short starting of point of nucelic acid that helps bind DNA to bind for DNA synthase
• In therumus polymerase to produce and stand DNA so single start are created

Reaction Steps in Electroporesis

• Start the separation to see the two chains and the hydrogen that breaks which is to 95 C
• Add a primer to go mix by the joining where there is a cooling at to 65 c that happens after they add
• After each chain is 72 c the bind which occurs for the production of the new copied DNA strand and is temp is carried for the cycle

Electroperhosis of Proteins and Gels

• Help used to analyze the DNA
• The samples are injected with electric field to track molecular movement
• The charge then impacts which direction in cathode and in anode as it changes to see to find rate
• In proteins for polymer and in DNA, are all used for gel tracking

Primers Role

• The Primer adheres to single DNA from to support DNA, for the pairing to begin and bind the DNA
• The primer does not let both strands rejoins

Additional info for primers

• If are the profile are same to suggest they had the same
• If the has similar properties is genetic

Additional Info for Proteins and gels

• Check with probe and ultraviolet helps with results
• Check position

Steps to Protein Test

• Gel, field, and electric current need
• Then can see how move
• Review numbers to see standard

Testing fragments

• To get from the blood by the to is transfer the results
• There a pH,
• The shorter time the better

Genomic testing

• Electrophoresis is different individuals and VNTRS from and unique genome
• Its help to track how far has genetic come and
• Its done by blood , saliva, hair
• PCR makes DNA found on gels
• Its gets produced is help to for the rate

Adding Electroporesis and Gels

• It happens in the cathode
• Then to maintain the PH

4/2/25 Part 5 Genomics

-Involve Genetic engineering, Electrophoresis, genetic screening

• For steps and sequencing in the process
• To discuss the gene edits and gene edits through the tool

Details on Gene Sequence

-They helps to form genes in a fragment

• To code a template so a primer helps helps
• In electro, it has multiple and all can be red,
• It cuts down and breaks bonds to help the template stay

Gene Edits Details

• Gene edits with code code 9's and is code is to add a to remove data as that happens with editing systems
• It codes the data bacteria need and defend
• Code segments code for data and viral DNA fragments

Other types of editting

-To focus with the based on the DNA it has active states

• Has parts the has information and allows bacteria to remember to pass

Gene Technology

Gene technology focuses on making protein and has stage called transoformation which involve transformation to add DNA or proteins to modified cells Plamsids help the process as they circle DNA, easy extract and have copy for data where there act as vector Promotes the RNA production as the help find the location for polymerases

Gene Technology

Gel tests are done to understand the code has been by the person unique profile The VNTRS and electro test that shows genetic code which can track any abnormalities or if data has been tempered with in a genome.

Adding in detail

• The samples need to bind and have DNA in electrophoresis which involves rays

##Gene Technology Bioinformatics provides analyze the sequence by seeing biological information from data They determine the pace for a genome and the data it entails

Adding

• To focus on to what is to be found

Gene Technology

Code in the human gene code is created that makes to focus on crisper case

Adding steps

• Primer code can cut genes and form DNA

Genetic engineering

• Engineering the ability to create new codes to have gene
• It knows where up to 20 base pair

Genetic engineering- Code 8

• It screens for cancer by the genetic, and help find early data,
• Help to to with early data for genetic
• Help see what is the best way to decide what is to done in future

Side Notes

• All code types may potential can cause disease
• Need for code help so it is easier to give and have better choice

Heterozygous

• With to understand how to make decision after or what is to decide about in future

Genetic engineering- Code 9

• It's important to look for to have the right decision the best options

Side steps

• To make data to see where what is correct or safe

##Genetic engineering- Code 10

• These types can hinder what does affect if for each type for example heart

Genetic engineering- Code 11

• The new gene and all be inherit and help

Genetic engineering- CrisPER-9

-In genetics one cannot interfere with the process, as the data is only about society and has right

• The ethical data helps to provide new cells and code to understand problems

Genetic engineering- GMO 22

• The steps are make the is insect free and to transfer into what in need
• These all affect some to survive and the climate

Step to Produce GMO-crop which is insect resistant

• To for genetic is the code for for the plant resistant and mix them