To Develop Botulinum Toxin E ) Insistant Genes?

Plasmids are small double stranded circular non chromosomal DNA molecules containing their own origin of replication. Hence, they are capable of replication independent of the chromosomal DNA in bacteria. Plasmids present in one or more copies per cell, can carry extra chromosomal DNA from one cell to another cell and serve as tools to clone and manipulate genes. Plasmids used exclusively for this purpose are known as vectors. The genes of interest can be inserted into these vector plasmids creating a recombinant plasmid. Recombinant plasmids can play a significant role in gene therapy, DNA vaccination, and drug delivery [Rapley, 2000].

One use of GFP is as a component of the plasmid pGLO. pGLO is made through recombinant DNA technology and consists of the GFP gene that codes for green fluorescence, a gene (bla) that codes for the enzyme beta-lactamase which produces bacteria resistance to the beta-lactam family of antibiotics of which ampicillin is one such antibiotic and lastly AraC which binds the arabinose thereby regulating expression of GFP. GFP will not be expressed unless the carbohydrate arabinose is present. Plasmid is small pieces of DNA that is distinct from a cell's chromosomal

Title Genetically transferring the GFP (green fluorescent protein) gene into E. Coli via heat shock Introduction Genetic transformation is the process by which desirable genetic material is extracted from one organism and forcibly placed in a different organism’s genetic complex. By adding foreign genes of organisms into different organisms, the genetic diversity of organisms is increasing exponentially and genetic transformation is one, if not the best, way to produce new alterations of genes as well as improve the functionality of other, already existing, genes. Although there are many different ways by which genetic transformation can be done, heat shock transformation was the method used in this lab. Heat shock is used to increase the

A cell suspension containing 0.5 -1.0 x 10^6 cells/ml in media 10% FBS (Fetal Bovine Serum) was placed on a 24- well plate so a monolayer can be created at the bottom of the wells. The first step will consist of the lab student aspirating the media from the wells making sure to disrupt the monolayer of cells at the bottom of the wells. Next the wells will be washed with PBS to remove dead cells and debris. After the wells are washed 1ml of media (DMEM) in a 5% concentration will be added to keep the cells from dehydrating. 5% Media is used because the goal here is to allow migration and minimize proliferation. Next the lab student will create a horizontal “scratch” along the bottom of the well using a P200 pipette. The lab student should use enough force to crate the scratch but not too much because too much force will create uneven scratches. Finally the treatments will be added to the wells. The plate will then be placed under the light microscope and pictures of the scratches will be taken which will be labeled as (T0) Time point zero. Other pictures will be taken 24 hrs. later at (T24) time point 24. The photos will then be analyzed and compared the lab group to determine if the is any cell

Caused by a 1 base pair deletion in SNAP-29 gene that results in the disappearance of a protein ( Horowitz, Lugassy, Rapaport, and Sprecher 2010)

For this part of the lab we used well-fed tetrhymena and 5% concentration of India ink throughout the experiment. The first two microcentrifuge tubes were positive and negative controls. The negative control contained only 0.2mL of the well-fed tetrahymena. The positive control contained 20 microliter of well-fed tetrahymena and 20 microliters of PFA in the microcentrifuge tube. We added 0.2mL of tetrahymena to the remaining centrifuge tubes. The third microcentrifuge tube (first condition) received 10 microliters of glucose. Then the fourth microcentrifuge tube received 10 microliter of colchicine (second condition). The fifth centrifuge was placed in an ice bath (so that the tetrhymena could be in a cold environment, also is the third condition).

The example given was doing at a much smaller scale than in the actual plant and was done with bottles, test tubes, and macaroni to represent the cells. Their was 7 steps between the upstream and downstream process that in an actual facility would take around 40 days. (FINISH THIS)

Genetic transformation is the process involving the insertion of new DNA into a bacteria cell such as E.Coli. While the inserted DNA contains a large chromosome, it also contains small circular pieces of DNA called plasmids. The injected plasmids hold specific genes as they are encoded through genetic engineering. Genetic engineering is described as “the direct manipulation of genes for practical purposes” (Urry et. al., 2016). The DNA injected directs the synthesis of proteins due to genetic expression and explains how bacteria is given the ability to resist antibiotics through the use of the pGLO plasmid. The “pGLO plasmid has been genetically engineered to carry the GFP gene which codes for the green fluorescent protein” (Biorad). This

Put the cells on the loop in the calcium chloride solution and the + plasma tube and spend the loop and the solution to dislodge the cell Mass

In genetic transformation, vectors are used to transfer genes from organism to another. In this experiment, we used plasmids as

Numerous standard laboratory techniques and instrumentation was used to obtain the forthcoming results. The ASAP1 expression plasmids were created by standard molecular biology methods, and then verified through cloned fragment sequencing. A plasmid was used for multiple reasons, the most prominent being that plasmids are very easy to manipulate to express a desired genetic element. In addition, they are very stable as well as self-replicating once initially constructed. HEK293A cells were used in experimentation and transfected with the ASAP1 plasmid. The HEK293A cells, which are very popular for transfection, were chosen as they are easy to reproduce, maintain, and have a high efficiency of transfection. HEK293A cells also have minimal processes that need to be accomplished in order to preform voltage-clamp experimentation. Transfection is simply the process by which

Plasmids are the extra-chromosomal DNA in which most bacteria contain in addition to their bacterial chromosome. Plasmids can be used as vectors to transfer DNA into a host bacterium. A nonpathogenic strain of Escherichia coli (E. coli) HB101;K-12, is the host bacterium used to take up the plasmid with the imported gene to be observed. pGLO is a recombinant plasmid that consists of a gene that codes for Green Fluorescent Protein (GFP) and also contains a gene, beta-lactam antibiotic (bla), that expresses antibiotic resistance. GFP is a protein that is produced by the jellyfish Aequoria victoria and has the ability to fluoresce green when exposed to ultraviolet (UV) light. The gene for GFP can be turned on in the transformed bacterial cell in the presence of the sugar, arabinose. The bacteria that contain the beta-lactam antibiotic have the ability to create beta-lactamase, which is an enzyme that hydrolyzes the beta-lactam rings of antibiotics which penicillin derivatives (among a broad range of others) contain, deeming them incompetent. Beta-lactam antibiotics inhibit the peptidoglycan layer of the bacterial cell wall and because E.coli is a gram-negative bacteria, its peptidoglycan layer is easily susceptible to inhibition.

Mark one tube “+” and the other “-”. The “-” tube will include the plasmid. Using a sterile graduated pipet, add 0.25 mL ice cold calcium chloride to each tube. Hold those tubes on ice. Obtain a starter plate of E. coli and using a sterile inoculating loop to transfer one large colony of bacteria form the starter plate to each tube of cold calcium chloride. In order to remove the bacteria from the transfer loop and break them apart, place the loop into the calcium chloride and twirl the loop rapidly. Dispose of the loop in a bio hazardous waste. Then, using a sterile inoculating loop, dip the loop into the DNA stock tube. When you remove the loop from the solution, check to be sure that there is a drop of liquid contained in the loop area. Transfer the liquid (approximately 10 μL to the “+” microfuge tube and twirl the loop to mix the plasmid into the solution. Place both the positive and negative tubes on ice for 15 minutes. While the tubes are on ice, obtain two Luria agar plates and two Luria agar plates with ampicillin. Label one Luria agar plate “+” and the other “-”; do the same for the Luria agar plates with ampicillin. After 15 minutes, remove the tubes from ice and immediately place them in a 42 degrees Celsius water bath for one minute. Remove the tubes from the water bath and immediately place them back on ice for two minutes. Remove them from the ice bath and add 0.25 mL of room temperature tryptic soy broth to each tube with a

Then added 200 ul of P1 Buffer (Red) to the tube and re-suspend pellet completely. We then centrifuged at 11 rpm for 1 min and discarded the supernatant. Then we added 200 ul of P2 Buffer (Green) and mix by inverting the tube 2 - 4 times. Cells are completely lysed when the solution appears clear, purple, and viscous. (Proceed to the next step within 1-2 minutes) We then centrifuged at 11 rpm for 1 min and discarded the supernatant. We added 400 ul of P3 Buffer (Yellow) and mix gently but thoroughly. The sample will turn yellow when the neutralization is complete then incubate the lysate at room temperature for 1-2 minutes. We then centrifuged at 11 rpm for 2 min and transferred the supernatant to a Zymo-Spin column. Once transferred we spun for 30 seconds at 11,00 rpm and then spun with 200 ul of Endo-wash for 30 seconds then 400 ul of plasmid wash buffer for a minute. After that we spun our column empty for an additional minute to remove an excess alcohol. We then transferred out column to a clean centrifuge tube and added 30 ul of Elution Buffer to the column and incubated at room temperature for a minute and centrifuged for 30 seconds. The result was our plasmid DNA.

The process of purifying DNA plasmid to express ΔK280 human tau was done in three steps: plasmid minipreps, electrophoresis, and sequencing.