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Homework answers / question archive / Ameer Taha Green Fluorescent Protein Bio 230 Professor Nonterah 4/18/2021 Abstract The aim of this experiment is to express the GFP in E
Ameer Taha Green Fluorescent Protein Bio 230 Professor Nonterah 4/18/2021 Abstract The aim of this experiment is to express the GFP in E.coli cells. the GFP gene. Transformation can be used to express GFP in E.coli cells, as a genetic engineering technique. During this step, international (pGLO) DNA will become E.coli and become E.coli. This plasmid contains the GFP gene, a gene encoding arabinose C proteins (araC), an arabinose promoter sequence encoding (PBad) and an integral oral sequence. In the presence of arabinose sugar, E Coli expresses the GFP protein if plasmid synthesis occurs. We can see that the cells are glowing under UV light. Two basic biological pathways can be used for this protein: expression report and bacterial localisation. Please use Hydrophobic Interaction Chromatography to perform purification and isolation of GFP (HIC). GFP is a very hydrophobic protein and thus HIC is used for separation and cleansing. We can see that the cells are glowing under UV light. Two basic biological pathways can be used for this protein: expression report and bacterial localisation. Please use Hydrophobic Interaction Chromatography to perform purification and isolation of GFP (HIC). GFP is a very hydrophobic protein and thus HIC is used for separation and cleansing. Background The genes considered to be responsible for the production of green fluorescent protein by fluorotec researchers. The 1962 green fluorescent protein was discovered by Shimomura and collaborators. Researchers also found that the green fluorescent protein and starfish species are related. They have discovered a plasmid which blocks a gene, called pGLO plasmid, since they discovered the starfish genus. A pGLO plasmid is a mutant organism with many chromosomes, such as the ampicillin and green fluorescent protein genes. As a result, the DNA of the pGLO plasmid has been designed to control the components of the arabinose operon. Furthermore, three enzymes are needed to produce the sugar arabinose, which are encoded by the araB, araA, and araD genes. Operon is located in front of the enzyme-coding genes. The promoter, which is the region of the DNA where RNA polymerase can begin to transcribe the genes that code for the enzymes, is the most essential part of the operon. These three enzymes, araB, araA, and araD, are part of the E.coli and help E.coli to digest sugar. The gene araC encodes PBAD, a DNA binding protein that is specific to the cell's DNA on the promoter. When E.coli cells absorb arabinose from their surroundings, it enters the cell. As E.coli joins the cells, it will respond with araC because it binds to DNA, and the form of araC will change. As a consequence, when binding occurs, RNA polymerases can transcribe the araB, araA, and araD genes, resulting in the synthesis of the three enzymes. The three enzymes then use arabiosis to complete it; because arabinose is no longer present, araC returns to its original status, and the RNA polymerase does not have to bind to the promoter anymore. The encoding process is then deactivated. Furthermore, araC is applied to the plasmid, however araB, A and D genes are used to code the starfish green fluorescent protein. AraC enhances the RNA polymerase required when arabinose is again present and translates and releases the green fluorescent protein. But where arabinose is not found it will not contain the green fluorescent protein and there will be no synthesis of proteins. The plasmid contains genes for beta-lactamase, indicating resistance to antibiotic ampicillin, in terms of the green fluorescent protein. Beta-lactamase contains the same plasmid that the green fluorescent protein will show the E.coli cells for the plasmid. However, the appropriate E. coli plasmid is referred to as a transformation, since the plasmid is only reduced in quantity. If the E. coli cell is in the cell and the cells are resistant to ampicillin in the medium growth area, this plasmid will have an ampicillin power. That is why. Because of the existence of ampicillin, E.coli cells will evolve. As a consequence, only plasmid-containing cells can develop and antibiotics kill cells without plasmids. The colour of the mature cells is fluorescent green when an arabinose is present and can be seen under UV light. If no arabinose is present, there is no growth, the ara operon is shut down, no green fluorescent protein produced, and the E. coli has a white creamy appearance. A protocol and measures need to be developed during the transition. The first step is to realize the CaCl2 cells. Ca+2 is used as a transitional solution for DNA to pass through and enter the cell wall. A thermal shock stage will then be used to enhance cell membrane DNA mobility in less time and use the transfer conditions. After that, the cells will grow for 10 minutes to express the protein tolerance to ampicillin. However, the antibiotic would have killed the cell if the cells had been plated in ampicillin before the addition of the experimental beta lactamase enzyme. This has led to the survival of transformed E.coli cells. Procedure We started the experiment with the transformation process. The first task was to designate two micro test tubes with the label +pGLO and -pGLO. Then we put them in a tube rack rack and added 250 meters of CaCl2, which is the transformation alternative, when opening the tube. Through a sterile transfer pipet, the solution was moved to test tubes. We placed the tubes in an ice-filled box afterwards. A single colony of bacteria, an agar plate, was separated using a sterile loop from the culture medium. When the +pGLO tube has been collected, we plunged the loop into the tube bottom and gently rotated it, so that the bacterial colony could spread into the processed solution. After placing the -pGLO tube in the icebox, the same protocol was performed. We found that when exposed to UV light, a green fluorescent light was released by the pGLO plasmid DNA solution.We removed a loop quantity of the pGLO solution and added it to the +pGLO tunnel by adding a new clean loop. After closing the vent, we returned it to the icebox. The tubes were incubated on ice for ten minutes, with the tubes fully immersed in ice. For 10 minutes, the markings of four agar plates with the LB/amp plate are awaited: LB/amp/ara: +pGLO, LB/amp plate: -pGLO After ten minutes, we shocked the tubes by immersing them for 50 seconds in a 42°C water bath. The tubes were then placed back on the ice for optimal transition efficiency. The tubes were incubated again for 2 minutes. Then we put the rack back on the table and we put in each tube 250 micrometers of LB nutrient broth and put the fresh pipette on the table and closed it again after the addition. After that, the tubes were incubated at room temperature for ten minutes. And we gently tapped the tubes, mixing them together. 100 microliters of the conversion and control suspensions in the required labels have been weighed and pipetted. The final step of the first day was to stack the plates, tap the plates, mark the stack at the end with the name of our party and place the stack in the incubator at 37° C upside down. Before the next laboratory, we wanted to abandon the stack. The first step on the next day was to remove the incubator from the transfer plate. We have two LB/amp/ara culture tubes in it and have the one + and the other - marked. We reached and immersed the green colony into the + labeling tube by a sterile circle. With the white colony we repeated the procedure, but used a clean loop and submerged it into the labelled tube this time. We then carefully turned the bolts around, closed the tubes and put them inside the incubator. The entire population was scattered. The tubes were held at 32° C overnight. The 2 mL of culture was transferred from the + tube to a + microtube the next day. The microtube was spun at full speed in the centrifuge for five minutes. After removing the supernatant and inspecting the pellet with a UV lamp, 250 microliters of TE solvent was added to the tube to fully suspend the pellet. We applied a lysozyme reduction and softly blended the tube material. Finally, the micro-tube has been put in the freezer and will be kept there until the next laboratory. In the next laboratory, we took the microtube from the freezer and thawed it with our fingertips. After that, the tube was centrifuged at full speed for 10 minutes. At the same time, we removed the cap and snapped off the bottom of the column prefilled with HIC, allowing the liquid buffers to drain, before applying the equilibrium buffer to the top of the column and draining to 1 mL. Before the next laboratory, the top and bottom were cut and held at room temperature. The tubing was quickly removed, and 250 microliters of + supernatant were transferred into a new micro-tube, called +, using a new pipet. It was mixed into the + supernatant with 250 microliters of connecting pipe and put in the freezer until the next lab session. In the following lab, we numbered three tubes (1–3) and lined them in a rack. Afterwards we took the column and removed it in tube number first before replacing it. Then, before moving it to the tube number 2, we pipetted 250 metric liters + supernatant on top of the column. The wash buffer has been applied to 250 microliters to the column. As the column began to leak, we moved it into the number third conduit, added 750 TE Buffer microliters and allowed the column to drain. The remaining 25 microliters were designated as "heat-treatments," while we took 25 microliters of the post-treatment tube, which we moved to a clean microcentrifuge tube labeled "native." Then we placed in each of the four tubes the same amount of sample buffer Laemmli. The tubes labeled "heat-treated" were heated at 95° C for five minutes. The samples were then placed in the wells and for the electrophoresis the gel box lid was closed. We turned on and for 30 minutes left the samples until electricity came to an end and the electrodes were disconnected from the gel cases. The gel was then removed gently from the frame and put into the staining tray prior to rinsing with . The gel was then put on a UV lightbox in a new plastic wrap. We took photos of them since we inspected lanes 6–10. We put 40 mL of Coomassie Blue stain into the tube, covered it and shaken it throughout the day after the gel has been inserted back into a strainer. After 24 hours the gel was stained overnight. Then, to determine the migration and create a normal curve to determine the molecular weight of the unknown proteins, we measured the distance between each standard and unknown protein bands. Data Transformation efficiency calculation: The number of colonies in the LB/amp/ara plate is 136 colonies. The concentration of DNA is 0.06 microgram/ microliter. The volume of pGLO used was 10 microliters. Total amount of DNA (microgram/ microliter) : 0.06 µg / µl * 10 µl = 0.6 µg of pGLO To find the volume of DNA spread on the plate, we add the 10 µl to the 500 µl of the nutrient broth and transformation solution: 10 + 500= 510 µl. Fraction of DNA used = (Volume spread on LB/amp/Ara plate)/ (Total Volume on test tube) Using 100 µl to put on each plate: 100/510 = 0.19608. To calculate how much DNA we spread on the plate: 0.6 µg * 0.19608 = 0.118 µg of pGLO spread on plate. Since the number of colonies present on the plate were 136 colonies. Transformation Efficiency: (total number of cells growing on agar plate / amount of DNA on agar plate). TE: 136 colonies / 0.118 µg = 1152.5 cells/µg Standard Curve Data: Graph: Gel Electrophoresis Results: Pre-stained gel (UV light) showing native GFP bands: Pre and Post- Pure GFP Concentration Calculations : Y= 0.0009x + 0.0114 X= ?−0.0114 0.0009 Pre purification GFP undiluted concentration X= 2.0−0.0114 0.0009 X= 2209.6 µg/ml Pre-purification GFP concentration 1:2 dilution → absorbance / y value = 0.657 ?= 0.657−0.0114 0.0009 X = 717.33 µg/ml X = 717 x 2 X = 1434.6 µg/ml Pre-purification GFP concentration 1:5 dilution → absorbance / y value = 0.196 ?= 0.196−0.0114 0.0009 → X = 205.11 µg/ml X = 205.11 x 5 →X = 1025.5 Post puri- GFP undiluted concentration → absorbance / y value = 0.974 X= 0.974−0.0114 0.0009 X= 1069.56 µg/ml Post-purification GFP concentration 1:2 dilution → absorbance / y value = 0.412 ?= 0.412−0.0114 0.0009 → X = 445.1 µg/ml X = 445.1 x 2 →X = 890.2 µg/ml Post-purification GFP concentration 1:5 dilution → absorbance / y value = 0.124 ?= 0.124−0.0114 0.0009 → X = 125.1 µg/ml X = 125.1 x 5 →X = 625.6 µg/ml Observations: The bacteria has evolved on the plate that contains only the LB nutrient broth. Under UV light, however, these bacteria do not shine a fluorescent violet. There is no bacterial development on the second layer, which also includes LB food broth and ampicillin. Bacterial development can be seen on the third dish, which includes LB nutrient broth, pGLO plasmid, and ampicillin. Under a UV light, however, these bacteria do not glow fluorescent violet. There are creamy white bacterial colonies on the fourth layer, which includes LB, pGLO, ampicillin, and arabinose. When exposed to UV radiation, these colonies turn fluorescent green. Discussion: The goal of this experiment is to observe the production of the GFP of E. coli which does not have the genes responsible for GFP manufacturing and to explore GFP features in order to decide whether the GFP can be used as a GE mark. We transformed the E. coli bacterium using the plasmid pGLO in optimal conditions in this particular experiment. In presence of high concentrated solution Ca2+ the chemical transition begins by making competent E. coli cells permeable to the membrane. The pGLO enters the bacterial cell after freezing and heating procedures. Four plates are prepared: LB-pGLO, LB/amp/pGLO, and LB/amp/amp/aras+pGLO. Four plates are prepared. The overall bacterial proliferation compared with the other plate in the first plate of LB+ ampicillin + pGLO + arabinose. The bacteria can not kill because of the beta-lactamase enzyme activated and activated with the addition of arabinose. Ara operon has been added to turn the Arabinose sugar and the GFP is generated by ara operon. The plate has also been lit under UV light, because arabinotic involvement triggers GFP gene expression. No bacterial growth in LB + plates. The ampicillin resistance gene is not expressed by these bacteria. The antibiotic destroys all the colonies. Furthermore, the bacteria are not exposed to pGLO and therefore the GFP gene cannot be expressed. Bacterial development decreased in LB + pGLO plates. Only a handful of bacteria survive and grow as beta-lactamase enzymes are expressed. As the GFP gene isn't activated by arabinoses, bacteria don't glisten under UV lamps to a fluorescent green. LB Plate: The proliferation of bacteria can be seen on the dish. These bacteria do not shine a green fluorescent color below the UV light without introduction to the pGLO plasmid that has the GFP gene. Transformation Efficiency means the capacity of the cell to absorb foreign DNA and to effectively express the DNA. The determined efficiency of transformation was 1152.5 cells/μg This indicates the number of bacteria in the pGLO plasmid that are able to efficiently express the gene. GFP was derived from the bacterial colonies that grew and could glow under UV light during extraction and purification. Added lysozyme, the cause of breakdown of the bacterial cell wall and destroying the bacteria. In conjunction with the GFP, the contents of bacteria are expelled from the cell. During the purification step, Hydrophobic Interaction Chromatography (HIC) distinguishes components of a mixture by hydrophobic type. The components are separated. GFP is a protein that is strongly hydrophobic. This facilitates separation with the HIC technique from other proteins. High levels of salt prevent binding of molecules to hydrophobic molecules, which is why the column is cleaned first with a buffer with a high salt content known as equilibrium. This would facilitate the washing of all hydrophobic proteins collected in the collection tube. The second wash was made with a reduced level of salt to wash out more proteins collected in the second tube. The last wash is performed by a zero-salt buffer Tris-EDTA, which makes the stored GFP in the third collection tube easier to wash. Therefore, if the third tube is placed under UV light, it has a fluorescent green tint. The SDS-PAGE technique distinguishes protein based on size and purety, which is based on polyacrylamide electrophoresis. This method is used for the purification of GFP. Electrophoresis gel is a method which, by size and load, separates the protein. In contrast to those of greater molecular weight, proteins with low molecular weight appear to migrate faster on the gel. The unpurified GFP samples display more bands because they contain more proteins of various molecular weights. We have replicated bands on every route, except the uniform routes, all approximately 33kD. In lanes 2-5 of the samples heated, the strips have a lighter hue than the original bands in lane 7-10. This is because heat denatures the proteins, thereby rendering them inert, by changing the secondary and tertiary structures. The absorbance of 6 identified GFP levels at a concentration of 595 nm is calculated for Bradford Assay. This is done to provide an equation for the pre-purified and post purified GFP sample concentration to be used. We did not, however, use all the six levels because the absorption value is 0,105 at 125 μg/ml and 0,950 at 1000 μg/ml. These values are below the same range, so the concentration is not determined. Y = 0.0009x + 0.0114 was the regular curve equation. The GFP 1:2 dilution and GFP 1:5 prepurification concentrations were 1434.6 μg/ml and 1025.5 respectively, respectively. Post-purified GFP concentration is 1069.56 μg/ml. Post-reinforced GFP solution concentration (1:2) was 890.2 μg/ml and the post-reinforced GFP solution concentration (1:5) was 625.6 μg/ml. Bradford test is non-protein-specific and unpurified sample concentrations are greater than predicted as it contains excess protein not removed from this test. Conclusion: This experiment was a success in general. Since we created GFP and investigated its molecular weight as one property, the project goals were met at the end. The data suggests promising results because the measurements were accurate and provided a clear picture of the whole protein. This project is also complicated since many approaches were used. References: https://www.youtube.com/watch?v=qK9aYnkIr3w https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3037093/ GRADING RUBRIC GFP DRAFT REPORT Report Component Poor Fair Average Abstract 0 Missing 0.5 Poorly written with many significant components missing 1 Includes most components but poorly written Introduction/ Purpose/Background (must include description of plasmid construct) 0 Missing 0.5 Introduction or Background missing; Missing info and discussion about plasmid construct Procedure 0 Procedure incomplete; major areas of procedure missing 0 Missing discussion of transformation process or data 0.5 Procedure written in notebook type detail rather than synopsis; some areas missing 0.5 Minimal information regarding purpose of plates with regard to results Calculation and explanation of 0 Missing calculation transformation frequency 0.5 Calculation present but poorly presented or inaccurate 1 Data tables & graphs poorly organized, some missing and missing components 1 Introduction or background Info about plasmid construct needs more information about genes other than GFP and their purpose 1 Procedure written as synopsis, but some areas are missing key info or are difficult to understand 1 Information about transformation process and plate data present; discussion of data is weak or incorrect interpretation 1 Calculation present but poorly presented; poor discussion 2 Most data tables & graphs present, organization could be improved, most components present 1 Both cell growth and extraction discussion present but inaccuracies exist 1 Purification is discussed but is missing details Discussion of transformation process, purpose of plate & analysis of data results Graphs and Tables (Data Presentation) 0 Significant amounts of data missing or data very poorly presented Discussion of cell growth and 0 Missing both extraction components 0.5 Missing discussion of either cell growth or extraction Discussion of column purification 0 Missing process and buffers in relation to visual aspects of process 0.5 little discussion of purification process Discussion of each sample and Bradford 0 Missing assay standard curve, calculations discussed including which dilutions most appropriate to use 0.5 errors in calculation or production of standard curve; no discussion of which dilutions used or why 1 standard curve accurate, but little discussion of dilution calculations Good 1.5 Overall complete and reasonably well written with some minor components missing 1.5 Most elements of the introduction, purpose & background are present 1.5 Most aspects of the project are covered and are clearly summarized 1.5 Transformation, purpose of plate and data are discussed but are missing a few key elements 1.5 Calculation present and well presented; discussion is weak 3 All data tables & graphs present, well organized; few elements missing 1.5 Both cell growth and extraction discussion present; discussion could be improved 1.5 Purification is discussed in relation to buffers and how they are used 1.5 Standard curve accurate, and some discussion of dilutions used and why Excellent 2 Well written with complete synopsis of report 2 Clearly introduces the project and overall goals; Complete info about plasmid construct, selection genes & other background information 2 Procedure covers all aspects of project, well written, organized and summarized 2 Transformation, purpose of plates and data results clearly discussed 2 Clear presentation of calculations and discussion of meaning 4 Data tables and graphs well organized, all components present 2 Cell growth and extraction discussion present, well written and complete 2 Purification is clearly discussed with references to observations made 2 Standard curve accurately presented and each sample correctly calculated and clearly discussed GRADING RUBRIC GFP DRAFT REPORT both pre and post purification protein 0 Not discussed assay samples discussed with regard to relationship and purity of GFP Gel data clearly presented including 0 Missing standard curve and calculations of molecular weights, including photo of gel with labels Discussion of pre and post purification 0 Not discussed lanes on gels with regard to comparison of overall purity of GFP Discussion of estimated molecular 0 Information not weight of GFP based on native protein included gel results Discussion of estimated molecular 0 Information not weight of GFP based on denatured included protein gel results Conclusions 0 Missing or so brief as to be missing Overall Organization of report 0 Report very poorly organized; sections jump around, many pieces of information missing or many repetitive sections Overall grammar /readability of report 0 Grammar is poor to the point of making the report unreadable Comments: 0.5 Minimal discussion of samples with regard to relationship & purity of GFP 0.5 Gel data poorly presented; missing photo or labels; little info on calculation of molecular weights 0.5 Minimal discussion of gel lanes with respect to purification effects 0.5 General reference to native protein gel results, but little discussion of data 1 Samples discussed, but not in relationship to pre & post purification differences 1 Gel data presented in reasonable form; some labeling or calculation information missing 1.5 Samples discussed with relationship to purification process 1 Gel lanes discussed but missing major comparisons that would add to understanding 1 Native gel bands discussed but lack details 1.5 Gel lanes discussed but missing minor elements that would help understanding 1.5 Native gel bands discussed and includes most significant elements of data discussion 0.5 General reference to denatured protein gel results but little discussion of data 1 Attempts made to discuss denatured protein gel results but discussion is incomplete, missing some significant elements 1.5 Discussion of estimated molecular weight present; few significant elements missing. 0.5 Conclusion is basically a repeat of data without clear significance of project results 1 poorly organized; sections jump around and do not tell a complete story; topics missing or many repetitive sections 1 Often uses inappropriate scientific terms 1 Conclusion has some data elements significance, others missing 1.5 Conclusion has description of significance of data elements but could be better organized 3 Overall organization of report is good, few areas need improvement (hard to understand or repetitive) 2 lacks organization; jumpy in some areas but can be followed with effort; some repetition of thoughts, small topics missing 2 Occasional inappropriate use of scientific terms 1.5 Gel data well presented; little information missing 3 Generally well written, but some grammar & spelling errors make for difficult comprehension 2 Pre & Post purification samples clearly discussed with regard to purification and purity of GFP 2 All gel data clearly presented; gel photo clearly labeled; sample calculations/data clearly presented 2 complete discussion of significance of bands on gels wrt to purification and purity of GFP 2 Discussion is complete, including clear reasoning for which band represents GFP and its estimated molecular weight based on standard curve 2 discussion complete, including clear reasoning for which band represents GFP and why & estimated molecular weight based on standard curve 2 Conclusion is well written, organized and clearly summarizes the major data/ideas from the project 4 Report well organized; information flows from one section to the next; clear and easy to follow and understand 4 Report well written, easy to understand; few grammar/spelling errors Ameer Taha Green Fluorescent Protein Bio 230 Professor Nonterah 4/18/2021 Abstract The aim of this experiment is to express the GFP in E.coli cells. the GFP gene. Transformation can be used to express GFP in E.coli cells, as a genetic engineering technique. During this step, international (pGLO) DNA will become E.coli and become E.coli. This plasmid contains the GFP gene, a gene encoding arabinose C proteins (araC), an arabinose promoter sequence encoding (PBad) and an integral oral sequence. In the presence of arabinose sugar, E Coli expresses the GFP protein if plasmid synthesis occurs. We can see that the cells are glowing under UV light. Two basic biological pathways can be used for this protein: expression report and bacterial localisation. Please use Hydrophobic Interaction Chromatography to perform purification and isolation of GFP (HIC). GFP is a very hydrophobic protein and thus HIC is used for separation and cleansing. We can see that the cells are glowing under UV light. Two basic biological pathways can be used for this protein: expression report and bacterial localisation. Please use Hydrophobic Interaction Chromatography to perform purification and isolation of GFP (HIC). GFP is a very hydrophobic protein and thus HIC is used for separation and cleansing. Background The genes considered to be responsible for the production of green fluorescent protein by fluorotec researchers. The 1962 green fluorescent protein was discovered by Shimomura and collaborators. Researchers also found that the green fluorescent protein and starfish species are related. They have discovered a plasmid which blocks a gene, called pGLO plasmid, since they discovered the starfish genus. A pGLO plasmid is a mutant organism with many chromosomes, such as the ampicillin and green fluorescent protein genes. As a result, the DNA of the pGLO plasmid has been designed to control the components of the arabinose operon. Furthermore, three enzymes are needed to produce the sugar arabinose, which are encoded by the araB, araA, and araD genes. Operon is located in front of the enzyme-coding genes. The promoter, which is the region of the DNA where RNA polymerase can begin to transcribe the genes that code for the enzymes, is the most essential part of the operon. These three enzymes, araB, araA, and araD, are part of the E.coli and help E.coli to digest sugar. The gene araC encodes PBAD, a DNA binding protein that is specific to the cell's DNA on the promoter. When E.coli cells absorb arabinose from their surroundings, it enters the cell. As E.coli joins the cells, it will respond with araC because it binds to DNA, and the form of araC will change. As a consequence, when binding occurs, RNA polymerases can transcribe the araB, araA, and araD genes, resulting in the synthesis of the three enzymes. The three enzymes then use arabiosis to complete it; because arabinose is no longer present, araC returns to its original status, and the RNA polymerase does not have to bind to the promoter anymore. The encoding process is then deactivated. Furthermore, araC is applied to the plasmid, however araB, A and D genes are used to code the starfish green fluorescent protein. AraC enhances the RNA polymerase required when arabinose is again present and translates and releases the green fluorescent protein. But where arabinose is not found it will not contain the green fluorescent protein and there will be no synthesis of proteins. The plasmid contains genes for beta-lactamase, indicating resistance to antibiotic ampicillin, in terms of the green fluorescent protein. Beta-lactamase contains the same plasmid that the green fluorescent protein will show the E.coli cells for the plasmid. However, the appropriate E. coli plasmid is referred to as a transformation, since the plasmid is only reduced in quantity. If the E. coli cell is in the cell and the cells are resistant to ampicillin in the medium growth area, this plasmid will have an ampicillin power. That is why. Because of the existence of ampicillin, E.coli cells will evolve. As a consequence, only plasmid-containing cells can develop and antibiotics kill cells without plasmids. The colour of the mature cells is fluorescent green when an arabinose is present and can be seen under UV light. If no arabinose is present, there is no growth, the ara operon is shut down, no green fluorescent protein produced, and the E. coli has a white creamy appearance. A protocol and measures need to be developed during the transition. The first step is to realize the CaCl2 cells. Ca+2 is used as a transitional solution for DNA to pass through and enter the cell wall. A thermal shock stage will then be used to enhance cell membrane DNA mobility in less time and use the transfer conditions. After that, the cells will grow for 10 minutes to express the protein tolerance to ampicillin. However, the antibiotic would have killed the cell if the cells had been plated in ampicillin before the addition of the experimental beta lactamase enzyme. This has led to the survival of transformed E.coli cells. Procedure We started the experiment with the transformation process. The first task was to designate two micro test tubes with the label +pGLO and -pGLO. Then we put them in a tube rack rack and added 250 meters of CaCl2, which is the transformation alternative, when opening the tube. Through a sterile transfer pipet, the solution was moved to test tubes. We placed the tubes in an ice-filled box afterwards. A single colony of bacteria, an agar plate, was separated using a sterile loop from the culture medium. When the +pGLO tube has been collected, we plunged the loop into the tube bottom and gently rotated it, so that the bacterial colony could spread into the processed solution. After placing the -pGLO tube in the icebox, the same protocol was performed. We found that when exposed to UV light, a green fluorescent light was released by the pGLO plasmid DNA solution.We removed a loop quantity of the pGLO solution and added it to the +pGLO tunnel by adding a new clean loop. After closing the vent, we returned it to the icebox. The tubes were incubated on ice for ten minutes, with the tubes fully immersed in ice. For 10 minutes, the markings of four agar plates with the LB/amp plate are awaited: LB/amp/ara: +pGLO, LB/amp plate: -pGLO After ten minutes, we shocked the tubes by immersing them for 50 seconds in a 42°C water bath. The tubes were then placed back on the ice for optimal transition efficiency. The tubes were incubated again for 2 minutes. Then we put the rack back on the table and we put in each tube 250 micrometers of LB nutrient broth and put the fresh pipette on the table and closed it again after the addition. After that, the tubes were incubated at room temperature for ten minutes. And we gently tapped the tubes, mixing them together. 100 microliters of the conversion and control suspensions in the required labels have been weighed and pipetted. The final step of the first day was to stack the plates, tap the plates, mark the stack at the end with the name of our party and place the stack in the incubator at 37° C upside down. Before the next laboratory, we wanted to abandon the stack. The first step on the next day was to remove the incubator from the transfer plate. We have two LB/amp/ara culture tubes in it and have the one + and the other - marked. We reached and immersed the green colony into the + labeling tube by a sterile circle. With the white colony we repeated the procedure, but used a clean loop and submerged it into the labelled tube this time. We then carefully turned the bolts around, closed the tubes and put them inside the incubator. The entire population was scattered. The tubes were held at 32° C overnight. The 2 mL of culture was transferred from the + tube to a + microtube the next day. The microtube was spun at full speed in the centrifuge for five minutes. After removing the supernatant and inspecting the pellet with a UV lamp, 250 microliters of TE solvent was added to the tube to fully suspend the pellet. We applied a lysozyme reduction and softly blended the tube material. Finally, the micro-tube has been put in the freezer and will be kept there until the next laboratory. In the next laboratory, we took the microtube from the freezer and thawed it with our fingertips. After that, the tube was centrifuged at full speed for 10 minutes. At the same time, we removed the cap and snapped off the bottom of the column prefilled with HIC, allowing the liquid buffers to drain, before applying the equilibrium buffer to the top of the column and draining to 1 mL. Before the next laboratory, the top and bottom were cut and held at room temperature. The tubing was quickly removed, and 250 microliters of + supernatant were transferred into a new micro-tube, called +, using a new pipet. It was mixed into the + supernatant with 250 microliters of connecting pipe and put in the freezer until the next lab session. In the following lab, we numbered three tubes (1–3) and lined them in a rack. Afterwards we took the column and removed it in tube number first before replacing it. Then, before moving it to the tube number 2, we pipetted 250 metric liters + supernatant on top of the column. The wash buffer has been applied to 250 microliters to the column. As the column began to leak, we moved it into the number third conduit, added 750 TE Buffer microliters and allowed the column to drain. The remaining 25 microliters were designated as "heat-treatments," while we took 25 microliters of the post-treatment tube, which we moved to a clean microcentrifuge tube labeled "native." Then we placed in each of the four tubes the same amount of sample buffer Laemmli. The tubes labeled "heat-treated" were heated at 95° C for five minutes. The samples were then placed in the wells and for the electrophoresis the gel box lid was closed. We turned on and for 30 minutes left the samples until electricity came to an end and the electrodes were disconnected from the gel cases. The gel was then removed gently from the frame and put into the staining tray prior to rinsing with . The gel was then put on a UV lightbox in a new plastic wrap. We took photos of them since we inspected lanes 6–10. We put 40 mL of Coomassie Blue stain into the tube, covered it and shaken it throughout the day after the gel has been inserted back into a strainer. After 24 hours the gel was stained overnight. Then, to determine the migration and create a normal curve to determine the molecular weight of the unknown proteins, we measured the distance between each standard and unknown protein bands. Data Transformation efficiency calculation: The number of colonies in the LB/amp/ara plate is 136 colonies. The concentration of DNA is 0.06 microgram/ microliter. The volume of pGLO used was 10 microliters. Total amount of DNA (microgram/ microliter) : 0.06 µg / µl * 10 µl = 0.6 µg of pGLO To find the volume of DNA spread on the plate, we add the 10 µl to the 500 µl of the nutrient broth and transformation solution: 10 + 500= 510 µl. Fraction of DNA used = (Volume spread on LB/amp/Ara plate)/ (Total Volume on test tube) Using 100 µl to put on each plate: 100/510 = 0.19608. To calculate how much DNA we spread on the plate: 0.6 µg * 0.19608 = 0.118 µg of pGLO spread on plate. Since the number of colonies present on the plate were 136 colonies. Transformation Efficiency: (total number of cells growing on agar plate / amount of DNA on agar plate). TE: 136 colonies / 0.118 µg = 1152.5 cells/µg Standard Curve Data: Graph: Gel Electrophoresis Results: Pre-stained gel (UV light) showing native GFP bands: Pre and Post- Pure GFP Concentration Calculations : Y= 0.0009x + 0.0114 X= ?−0.0114 0.0009 Pre purification GFP undiluted concentration X= 2.0−0.0114 0.0009 X= 2209.6 µg/ml Pre-purification GFP concentration 1:2 dilution → absorbance / y value = 0.657 ? = 0.657−0.0114 0.0009 X = 717.33 µg/ml X = 717 x 2 X = 1434.6 µg/ml Pre-purification GFP concentration 1:5 dilution → absorbance / y value = 0.196 ? = 0.196−0.0114 0.0009 → X = 205.11 µg/ml X = 205.11 x 5 →X = 1025.5 Post puri- GFP undiluted concentration → absorbance / y value = 0.974 X= 0.974−0.0114 0.0009 X= 1069.56 µg/ml Post-purification GFP concentration 1:2 dilution → absorbance / y value = 0.412 ? = 0.412−0.0114 0.0009 → X = 445.1 µg/ml X = 445.1 x 2 →X = 890.2 µg/ml Post-purification GFP concentration 1:5 dilution → absorbance / y value = 0.124 ? = 0.124−0.0114 0.0009 → X = 125.1 µg/ml X = 125.1 x 5 →X = 625.6 µg/ml Observations: The bacteria has evolved on the plate that contains only the LB nutrient broth. Under UV light, however, these bacteria do not shine a fluorescent violet. There is no bacterial development on the second layer, which also includes LB food broth and ampicillin. Bacterial development can be seen on the third dish, which includes LB nutrient broth, pGLO plasmid, and ampicillin. Under a UV light, however, these bacteria do not glow fluorescent violet. There are creamy white bacterial colonies on the fourth layer, which includes LB, pGLO, ampicillin, and arabinose. When exposed to UV radiation, these colonies turn fluorescent green. Discussion: The goal of this experiment is to observe the production of the GFP of E. coli which does not have the genes responsible for GFP manufacturing and to explore GFP features in order to decide whether the GFP can be used as a GE mark. We transformed the E. coli bacterium using the plasmid pGLO in optimal conditions in this particular experiment. In presence of high concentrated solution Ca2+ the chemical transition begins by making competent E. coli cells permeable to the membrane. The pGLO enters the bacterial cell after freezing and heating procedures. Four plates are prepared: LB-pGLO, LB/amp/pGLO, and LB/amp/amp/aras+pGLO. Four plates are prepared. The overall bacterial proliferation compared with the other plate in the first plate of LB+ ampicillin + pGLO + arabinose. The bacteria can not kill because of the beta-lactamase enzyme activated and activated with the addition of arabinose. Ara operon has been added to turn the Arabinose sugar and the GFP is generated by ara operon. The plate has also been lit under UV light, because arabinotic involvement triggers GFP gene expression. No bacterial growth in LB + plates. The ampicillin resistance gene is not expressed by these bacteria. The antibiotic destroys all the colonies. Furthermore, the bacteria are not exposed to pGLO and therefore the GFP gene cannot be expressed. Bacterial development decreased in LB + pGLO plates. Only a handful of bacteria survive and grow as beta-lactamase enzymes are expressed. As the GFP gene isn't activated by arabinoses, bacteria don't glisten under UV lamps to a fluorescent green. LB Plate: The proliferation of bacteria can be seen on the dish. These bacteria do not shine a green fluorescent color below the UV light without introduction to the pGLO plasmid that has the GFP gene. Transformation Efficiency means the capacity of the cell to absorb foreign DNA and to effectively express the DNA. The determined efficiency of transformation was 1152.5 cells/μg This indicates the number of bacteria in the pGLO plasmid that are able to efficiently express the gene. GFP was derived from the bacterial colonies that grew and could glow under UV light during extraction and purification. Added lysozyme, the cause of breakdown of the bacterial cell wall and destroying the bacteria. In conjunction with the GFP, the contents of bacteria are expelled from the cell. During the purification step, Hydrophobic Interaction Chromatography (HIC) distinguishes components of a mixture by hydrophobic type. The components are separated. GFP is a protein that is strongly hydrophobic. This facilitates separation with the HIC technique from other proteins. High levels of salt prevent binding of molecules to hydrophobic molecules, which is why the column is cleaned first with a buffer with a high salt content known as equilibrium. This would facilitate the washing of all hydrophobic proteins collected in the collection tube. The second wash was made with a reduced level of salt to wash out more proteins collected in the second tube. The last wash is performed by a zero-salt buffer Tris-EDTA, which makes the stored GFP in the third collection tube easier to wash. Therefore, if the third tube is placed under UV light, it has a fluorescent green tint. The SDS-PAGE technique distinguishes protein based on size and purety, which is based on polyacrylamide electrophoresis. This method is used for the purification of GFP. Electrophoresis gel is a method which, by size and load, separates the protein. In contrast to those of greater molecular weight, proteins with low molecular weight appear to migrate faster on the gel. The unpurified GFP samples display more bands because they contain more proteins of various molecular weights. We have replicated bands on every route, except the uniform routes, all approximately 33kD. In lanes 2-5 of the samples heated, the strips have a lighter hue than the original bands in lane 7-10. This is because heat denatures the proteins, thereby rendering them inert, by changing the secondary and tertiary structures. The absorbance of 6 identified GFP levels at a concentration of 595 nm is calculated for Bradford Assay. This is done to provide an equation for the pre-purified and post purified GFP sample concentration to be used. We did not, however, use all the six levels because the absorption value is 0,105 at 125 μg/ml and 0,950 at 1000 μg/ml. These values are below the same range, so the concentration is not determined. Y = 0.0009x + 0.0114 was the regular curve equation. The GFP 1:2 dilution and GFP 1:5 prepurification concentrations were 1434.6 μg/ml and 1025.5 respectively, respectively. Post-purified GFP concentration is 1069.56 μg/ml. Post-reinforced GFP solution concentration (1:2) was 890.2 μg/ml and the post-reinforced GFP solution concentration (1:5) was 625.6 μg/ml. Bradford test is non-protein-specific and unpurified sample concentrations are greater than predicted as it contains excess protein not removed from this test. Conclusion: This experiment was a success in general. Since we created GFP and investigated its molecular weight as one property, the project goals were met at the end. The data suggests promising results because the measurements were accurate and provided a clear picture of the whole protein. This project is also complicated since many approaches were used. References: https://www.youtube.com/watch?v=qK9aYnkIr3w https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3037093/ Abstract The aim of this experiment is to express the GFP in E.coli cells. The GFP gene. Transformation can be used to express GFP in E.coli cells, as a genetic engineering technique. During this step, international (PGLO) DNA will become E.coli and become E.coli. This plasmid contains the GFP gene, a gene ehcoandarasnose er proteins Gertrude Nonterah (araC), an arabinose promoter sequence encoding (PBad) and an integral oral international? The PGLO does not become E. coli. It is transformed into the E coli cells. sequence. In the presence of arabinose sugar, E Coli expresses the GFP protein if plasmid synthesis occurs. We can see that the cells are glowing under UV light. Two basic biological pathways can be used for this protein: expression report and bacterial localisation. Gertrude Nonterah if the plasmid successfully enters the bacteria Please use Hydrophobic Interaction Chromatography to perform purification and isolation of GFP (HIC). GFP is a very hydrophobic protein and thus HIC is used for separation and cleansing. Gertrude Nonterah We can see that the cells are glowing under UV light. Two basic biological pathways can be used for this protein: expression report and bacterial localisation. integral oral sequence? I don't understand this. Please use Hydrophobic Interaction Chromatography to perform purification and isolation of GFP (HIC). GFP is a very hydrophobic protein and thus HIC is used for separation and cleansing. Gertrude Nonterah This abstract is not well written. You ant to provide a summary of what was done. And it should be written in past tense.Look at the examples of the abstracts | provided in Canvas to write a better abstract. Background In an abstract, you are not giving instructions. You are writing about what you did. The genes considered to be responsible for the production of green fluorescent protein by fluorotec researchers. The 1962 green fluorescent protein was discovered by Shimomura and collaborators. Researchers also found that the green fluorescent protein and starfish species are related. They have discovered a plasmid which blocks a Gertrude Nonterah Did they find that? Jellyfish and starfish are different species. gene, called PGLO plasmid, since they discovered the starfish genus. A PGLO plasmid is a mutant organism with many chromosomes, such as the ampicillin and green fluorescent protein genes. As a result, the DNA of the PGLO plasmid has been Gertrude Nonterah designed to control the components of the arabinose operon. Furthermore, three enzymes are needed to produce the sugar arabinose, which are encoded by the arab, ??? araA, and araD genes. Operon is located in front of the enzyme-coding genes. The promoter, which is the region of the DNA where RNA polymerase can begin to Gertrude Nonterah transcribe the genes that code for the enzymes, is the most essential part of the operon. These three enzymes, arab, araA, and arad, are part of the E.coli and help E.coli to PGLO is a plasmid - a circular piece of DNA and not a mutant organism. Please read the lab again to learn what the PGLO plasmid really is. digest sugar. The gene araC encodes PBAD, a DNA binding protein that is specific to the cell's DNA on the promoter. When E.coli cells absorb arabinose from their surroundings, it enters the cell. As E.coli joins the cells, it will respond with arac because it binds to DNA, and the form of araC will change. As a consequence, when binding occurs, RNA polymerases can transcribe the arab, araA, and araD genes, resulting in the synthesis of the three enzymes. The three enzymes then use arabiosis to complete it; because arabinose is no longer present, araC returns to its original status, and the RNA polymerase does not have to bind to the promoter anymore. The encoding process is then deactivated. Furthermore, araC is applied to the plasmid, however araB, A and D genes are used to code the starfish green fluorescent protein. Aroranbonco the DNA olumaroc roured when arabinocicoin procent and AraC enhances the RNA polymerase required when arabinose is again present and translates and releases the green fluorescent protein. But where arabinose is not found it will not contain the green fluorescent protein and there will be no synthesis of proteins. The plasmid contains genes for beta-lactamase, indicating resistance to antibiotic ampicillin, in terms of the green fluorescent protein. Beta-lactamase contains the same plasmid that the green fluorescent protein will show the E.coli cells for the plasmid. However, the appropriate E. coli plasmid is referred to as a transformation, since the plasmid is only reduced in quantity. If the E. coli cell is in the cell and the cells are resistant to ampicillin in the medium growth area, this plasmid will have an ampicillin power. That is why. Because of the existence of ampicillin, E.coli cells will evolve. As a consequence, only plasmid-containing cells can develop and antibiotics kill cells without plasmids. The colour of the mature cells is fluorescent green when an arabinose is present and can be seen under UV light. If no arabinose is present, there is no growth, the ara operon is shut down, no green fluorescent protein produced, and the E. coli has a white creamy appearance. A protocol and measures need to be developed during the transition. The first step is to realize the CaCl2 cells. Ca+2 is used as a transitional solution for DNA to pass through and enter the cell wall. A thermal shock stage will then be used to enhance cell membrane DNA mobility in less time and use the transfer conditions. After that, the cells will grow for 10 minutes to express the protein tolerance to ampicillin. However, the antibiotic would have killed the cell if the cells had been plated in ampicillin before the addition of the experimental beta lactamase enzyme. This has led to the survival of transformed E.coli cells. Please also talk briefly about the purification and SDS-PAGE. This background is not well written at all. A lot scientific inaccuracies make for difficult comprehension. Please take the time to understand what the PGLO plasmid is and the genes in the plasmid Procedure Gertrude Nonterah We started the experiment with the transformation process. The first task was to designate two micro test tubes with the label +PGLO and -pGLO. Then we put them in a make sure to be consistent and write the WHOLE procedure in past tense. tube rack rack and added 250 meters of CaCl2, which is the transformation alternative, when opening the tube. Through a sterile transfer pipet, the solution was moved to test tubes. We placed the tubes in an ice-filled box afterwards. A single colony of bacteria, Gertrude Nonterah an agar plate, was separated using a sterile loop from the culture medium. When the microliters. Not meters. +PGLO tube has been collected, we plunged the loop into the tube bottom and gently rotated it, so that the bacterial colony could spread into the processed solution. After placing the - GLO tube in the icebox, the same protocol was performed. We found that when exposed to UV light, a green fluorescent light was released by the PGLO plasmid DNA solution. We removed a loop quantity of the PGLO solution and added it to the +PGLO tunnel by adding a new clean loop. After closing the vent, we returned it to the icebox. The tubes were incubated on ice for ten minutes, with the tubes fully immersed in ice. For 10 minutes, the markings of four agar plates with the LB/amp plate are awaited: LB/amp/ara: +pGLO, LB/amp plate: -OGLO After ten minutes, we shocked the tubes by immersing them for 50 seconds in a 42°C water bath. The tubes were then placed back on the ice for optimal transition efficiency. The tubes were incubated again for 2 minutes. Then we put the rack back on the table and we put in each tube 250 micrometers of LB nutrient broth and put the fresh pipette on the table and closed it again after the addition. After that, the tubes were incubated at room temperature for ten minutes. And we gently tapped the tubes, mixing them together. 100 microliters of the GFP Transformation Data -PGLO LB Confluent bacterial grown over entire plate surface -PGLO LB amp No bacterial growth on plate +PGLO LB amp 73 colonies; Colonies are creamy white color under normal light; Colonies do not fluoresce under UV light +PGLO LB amp ara 136 colonies; Colonies are creamy white color under normal light; colonies fluoresce bright green under UV light - DNA LBIAMP DNA LB/AMP LBIAMPIARA Transformation efficiency calculation: The number of colonies in the LB/amp/ara plate is 136 colonies. The concentration of DNA is 0.06 microgram/ microliter. The volume of PGLO used was 10 microliters. Total amount of DNA (microgram/ microliter): 0.06 ug/ul * 10 pl = 0.6 ug of PGLO To find the volume of DNA spread on the plate, we add the 10 ul to the 500 ul of the nutrient broth and transformation solution: 10 + 500= 510 ul. Fraction of DNA used = (Volume spread on LB/amp/Ara plate) (Total Volume on test tube) Fraction of DNA used = (Volume spread on LB/amp/Ara plate) (Total Volume on test tube) Using 100 pl to put on each plate: 100/510 = 0.19608. To calculate how much DNA we spread on the plate: 0.6 ug * 0.19608 = 0.118 ug of PGLO spread on plate. Since the number of colonies present on the plate were 136 colonies. Transformation Efficiency: (total number of cells growing on agar plate / amount of DNA on agar plate). TE: 136 colonies / 0.118 ug = 1152.5 cells/pg Standard Curve Data: Concentration(ug/ml) 1000 750 500 375 250 125 Pre Pure GFP undiluted Pre pure GFP 1:2 dilution Pre pure GFP 1:5 dilution Post-Pure GFP undiluted Post-Pure GFP 1:2 dilution Post-Pure GFP 1:5 dilution As95 0.930 0.701 0.470 0.350 0.245 0.105 >2.0 0.657 0.196 0.974 0.412 0.124 Graph: Absorbance at 595 nm of 4 conectrations of GFP solutions 0.8 0.7 y = 0.0009x +0.0114 R = 0.9996 0.6 0.5 Absorbance at 595 nm 0.4 0.3 0.2 0.1 0 0 100 200 300 400 500 600 700 800 Concentration(ng/ml) Gel Electrophoresis Results: LLULO label your gels 50kd 37kd 25kd 15ka 10kd Pre-stained gel (UV light) showing native GFP bands: Pre and Post-Pure GFP Concentration Calculations : Y= 0.0009x + 0.0114 X= Y-0.0114 0.0009 Pre purification GFP undiluted concentration X= 2.0-0.0114 0.0009 since the number is greater than 2, you cannot assume a value and thus you cannot use it in a calculation. X= 2209.6 pg/ml Pre-purification GFP concentration 1:2 dilution + absorbance /y value = 0.657 X = 0.657-0.0114 0.0009 X = 717.33 pg/ml X = 717 x 2 X = 1434.6 pg/ml Pre-purification GFP concentration 1:5 dilution → absorbance / y value = 0.196 X = 0.196-0.0114 0.0009 X = 205.11 ug/ml X = 205.11 x 5X = 1025.5 Post puri- GFP undiluted concentration → absorbance / y value = 0.974 X= 0.974-0.0114 0.0009 X= 1069.56 ug/ml Post-purification GFP concentration 1:2 dilution → absorbance /y value = 0.412 X = 0.412-0.0114 0.0009 X = 445.1 pg/ml X = 445.1 x 2 X = 890.2 pg/ml Post-purification GFP concentration 1:5 dilution → absorbance /y value = 0.124 0.124-0.0114 X = 0.0009 X = 125.1 pg/ml X = 125.1 x 5 X = 625.6 pg/ml Observations: The bacteria has evolved on the plate that contains only the LB nutrient broth. Under Gertrude Nonterah Include this in your discussion since these were not the only observations made. UV light, however, these bacteria do not shine a fluorescent violet. There is no bacterial development on the second layer, which also includes LB food broth and ampicillin. Bacterial development can be seen on the third dish, which includes LB nutrient broth, PGLO plasmid, and ampicillin. Under a UV light, however, these bacteria do not glow Gertrude Nonterah fluorescent violet. There are creamy white bacterial colonies on the fourth layer, which includes LB, PGLO, ampicillin, and arabinose. When exposed to UV radiation, these Wrong term. The bacteria did not "evolve". colonies turn fluorescent green. Why did you see these results? Refer back to our Discussion: discussions to understand WHY these we the results we saw. The goal of this experiment is te ebserve the production of the GFP ef E.coli which does Gertrude Nonterah not have the genes responsible for GFP manufacturing and to explore GFP features in order to decide whether the GFP can be used as a GE mark. We transformed the E. coli bacterium using which second layer? Please use the correct terminology and don't make stuff up. Also please say "bacterial growth" not "bacterial development". [...] the plasmid PGLO in optimal conditions in this particular experiment. In presence of high concentrated solution Ca2+ the chemical transition begins by making competent E. coli cells permeable to the membrane. The PGLO enters the bacterial cell after freezing and heating Gertrude Nonterah procedures. Four plates are prepared: LB-pGLO, LB/amp/pGLO, and LB/amp/amp/aras+pGLO. ?? What is a GE mark? Four plates are prepared. The overall bacterial proliferation compared with the other plate in the first plate of LB+ ampicillin + PGLO + arabinose. The bacteria can not kill because of the beta-lactamase enzyme Gertrude Nonterah what were the optimal conditions? 1 1 More Comment : Gertrude Nonterah 1 More Comment Gertrude Nonterah activated and activated with the addition of arabinose. Ara operon has been added to turn the Arabinotic?? Arabinose sugar and the GFP is generated by ara operon. The plate has also been lit under UV light, because arabinotic involvement triggers GFP gene expression. No bacterial growth in LB + plates. The ampicillin resistance gene is not expressed by Gertrude Nonterah these bacteria. The antibiotic destroys all the colonies. Furthermore, the bacteria are not exposed to PGLO and therefore the GFP gene cannot be expressed. Please look at the data again. There was confluent growth of bacteria on the LB plates. So saying there was no growth is incorrect. Bacterial development decreased in LB + PGLO plates. Only a handful of bacteria survive and grow as beta-lactamase enzymes are expressed. As the GFP gene isn't activated by arabinoses, bacteria don't glisten under UV lamps to a fluorescent green. LB Plate: The proliferation of bacteria can be seen on the dish. These bacteria do not shine a green fluorescent Gertrude Nonterah glow NOT glisten color below the UV light without introduction to the PGLO plasmid that has the GFP gene. Transformation Efficiency means the capacity of the cell to absorb foreign DNA and to effectively express the DNA. The determined efficiency of transformation was 1152.5 cells/ug Gertrude Nonterah This indicates the number of bacteria in the PGLO plasmid that are able to efficiently express the gene. GFP was derived from the bacterial colonies that grew and could glow under UV light during extraction and purification. Added lysozyme, the cause of breakdown of the bacterial cell there is no need to include what you wrote in the procedure in your discussion. You do this in several parts of your discussion. Be min of this. [...] wall and destroying the bacteria. In conjunction with the GFP, the contents of bacteria are expelled from the cell. During the purification step, Hydrophobic Interaction Chromatography (HIC) distinguishes components of a mixture by hydrophobic type. The components are separated. GFP is a protein that is strongly hydrophobic. This facilitates separation with the HIC technique from other proteins. High levels of salt prevent binding of molecules to hydrophobic molecules, which is why the column is cleaned first with a buffer with a high salt content known as equilibrium. This would facilitate the washing of all hydrophobic proteins collected in the collection tube. The second wash was made with a reduced level of salt to wash out more proteins collected in the second tube. The last wash is performed by a zero-salt buffer Tris-EDTA, which makes the stored GFP in the third collection tube easier to wash. Therefore, if the third tube is placed under UV light, it has a fluorescent green tint. The SDS-PAGE technique distinguishes protein based on size and purety, which is based on polyacrylamide electrophoresis. This method is used for the purification of GFP. Electrophoresis gel is a method which, by size and load, separates the protein. In contrast to those of greater molecular weight, proteins with low molecular weight appear to migrate faster on the gel. The unpurified GFP samples display more bands because they contain more proteins of various molecular weights. We have replicated bands on every route, except the uniform routes, all approximately 33kD. In lanes 2-5 of the samples heated, the strips have a lighter hue than the original bands in lane 7-10. This is because heat denatures the proteins, thereby rendering them inert, by changing the secondary and tertiary structures. The absorbance of 6 identified GFP levels at a concentration of 595 nm is calculated for Bradford Assay. This is done to provide an equation for the pre-purified and post purified GFP sample concentration to be used. We did not, however, use all the six levels because the absorption value is 0,105 at 125 ug/ml and 0,950 at 1000 ug/ml. These values are below the same range, so the concentration is not determined. Y = 0.0009x + 0.0114 was the regular curve equation. The GFP 1:2 dilution and GFP 1:5 prepurification concentrations were 1434.6 ug/ml and 1025.5 respectively, respectively. Post-purified GFP concentration is 1069.56 ug/ml. Post-reinforced GFP solution concentration (1:2) was 890.2 ug/ml and the post-reinforced GFP solution concentration (1:5) was 625.6 ug/ml. Bradford test is non-protein-specific and unpurified sample concentrations are greater than predicted as it contains excess protein not removed from this test. Which dilution is best? How did the purification process affect the concentrations pre- and post-purification? Which dilution is best? How did the purification process affect the concentrations pre- and post-purification? Conclusion: This experiment was a success in general. Since we created GFP and investigated its molecular weight as one property, the project goals were met at the end. The data suggests promising results because the measurements were accurate and provided a clear picture of the whole protein. This project is also complicated since many approaches were used. Restate the what we learned about this GFP from starfish i.e the concentration and the protein size References: and how that relates back to our aims. https://www.youtube.com/watch?v=qK9aYnkIr3w https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3037093/
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