Bacterial Transformation Efficiency: E.Coli with pGLO

Bacterial Transformation Efficiency: E.Coli with pGLO Bacterial Transformation Efficiency in E.Coli with pGLO Plasmids By: Richard Stone Introduction The conversion of one genotype into another by the introduction of exogenous DNA (that is, bits of DNA from an external source) is termed transformation. The transformation was discovered in Streptococcus pneumoniae in 1928 by Frederick Griffith; in 1944, Oswald T. Avery, Colin M. MacLeod, and Maclyn McCarty demonstrated that the transforming principle was DNA. Both results are milestones in the elucidation of the molecular nature of genes. 1 Bacteria transformation is the process of a bacteria absorbing and expressing foreign genetic information using plasmids. Plasmids are small circular molecules of DNA that holds a small number of genes. The plasmids used in the experiment have the ampicillin resistance gene. Ampicillin (amp) is an antibiotic used to kill bacteria such as E. coli, the bacteria used in the experiment. E. coli (Escherichia coli) is a simple bacterium commonly found in our bodys and in everyday life but most commonly found in mammals intestines. Glowing Fluorescent Proteins (GFPs) is the gene found in jellyfish that holds bioluminescent properties and glow under UV light. By knowing the location of the gene, scientists can cut out the GFP gene from the jellyfish DNA. They do this using restriction enzymes to which recognize and cut DNA in a specific region of nucleotides to acquire a specific gene. Once the gene is isolated, it can be used in the experiment and glued into a plasmid that contains the AMP gene. This is done by sticky ends as the Jellyfish DNA binds to the amp resistance plasmid using hydrogen bonds which are hen sealed by DNA ligase. This creates pGLO a plasmid which is used in the experiment in the transformation of the bacteria. Before it can be part of the transformation the bacteria must be made competent to accept the pGLO. This is done by heat shocking the bacteria which makes it easier for the pGLO to be incorporated into the bacteria. For the bacteria to fluoresce sunder UV light it must be in presence of arabinose sugars, which turns on the gene for the production of Glowing Fluorescent Proteins.2 The amp Resistance gene enables bacteria to survive in the presence of the antibiotic ampicillin. When a plasmid containing both the GFP gene and AMP gene (pGLO) is transferred into an E. coli bacterium, the transformed cells can be grown in a culture dish that contains ampicillin. Only a small number of bacteria cells will be transformed and grow on the LB (lysoge ny broth) and amp plates and glow. 3 The experiment demonstrates how Bacteria is modified to express a specific gene through the process of bacterial transformation. The purpose of this experiment is to find the efficiency of bacterial transformation in E. Coli bacteria by observing their expression of the plasmids. This is calculated by determining the frequency of the bacterium with GFPs and arabinose sugars by counting the glowing colonies. It was the results for each plate was hypothesized before the experiment. The LB plate with only the bacteria and no pGLO administered will grow a lawn of bacteria and have no glowing properties. The LB with ampicillin but bacteria without pGLO will not survive at all and there will be no bacteria growth. The LB plate with amp and bacteria with the pGLO will have bioluminescent properties but only a very small percentage of the bacteria will survive the amp and bacterial transformation will occur. Finally, the LB with no amp but the bacteria with the pGLO will form a lawn of bacteria and the bacteria that is transformed will glow like the previous plate. The efficiency of the bacterial transformation is hypothesized using in class discussion and background knowledge, to be about 810-4 %. 4 Materials and Methods E. coli bacteria cultures 100-1000  µl micropipette 0.5-10  µl micropipette sterile tips 2 sterile 15-ml test tubes 500 ÃŽÂ ¼L of ice cold 0.05M CaCl2 (ph. 6.1) 500 ÃŽÂ ¼L of lysogeny broth/agar a spreading rod Bunsen burner 4 agar plates: 2 ampicillins+ and 2 ampicillin an incubator a sterile inoculating loop 10 ÃŽÂ ¼L of pAMP solution a timer ice tape sterile glass beads a water bath 1. Use a permanent marker to label one sterile 15-ml tube +, and another -. 2. Use a 100-1000  µl micropipette and sterile tip to add 250  µl of CaCl2 (calcium chloride) solution to each tube. 3. Place both tubes on ice. 4. Use a sterile inoculating loop to transfer a visible mass of E. coli from a starter plate to the + tube: a. Sterilize loop in Bunsen burner flame until it glows red hot. b. Carefully, stab loop into agar to cool. c. Scrape up a visible mass of E. coli, but be careful not to transfer any agar. (Impurities in agar can inhibit transformation.) d. Immerse loop tip in CaCl2 solution and vigorously tap against the wall of the tube to dislodge bacteria. Hold tube up to light to observe the bacteria drop off into the calcium chloride solution. Make sure cell mass is not left on a loop or on side of tube. e. Sterilize loop before setting it on the lab bench. 5. Immediately suspend cells in the + tube by repeatedly pipetting in and out, using a 100-1000  µl micropipette with a fresh sterile tip.a. Pipet carefully to avoid making bubbles in suspension or splashing suspension far up sides of the tube. b. Hold tube up to light to check that suspension is homogeneous. No visible clumps of cells should remain. 6. Return + tube to ice. 7. Transfer the second mass of cells to tube as described in Step 4, and resuspend cells as described in Step 5. 8. Return tube to ice. Both tubes should be on the ice. 9. Use a 0.5-10  µl micropipette to add 10  µl of 0.005  µg/ µl pGFP solution directly into cell suspension in the + tube. Tap tube with a finger to mix. Avoid making bubbles in suspension or splashing suspension up to the sides of the tube. [DO NOT ADD pGFP TO THE - TUBE.] 10. Return + tube to ice. Incubate both tubes on ice for 15 minutes. 11. While cells are incubating, use a permanent marker to label two LB plates and two LB/amp plates with name and the date. Label one LB/amp plate + GFP. This is the experimental plate. Label the other LB/amp plate - GFP. This is a negative control. Label one LB plate + GFP. This is a positive control. Label the other LB plate - GFP. This is a negative control. 12. Following the 15-minute incubation on ice, heat shock the cells in both the + and tubes. It is critical that cells receive a sharp and distinct shock: a. Carry ice beaker to the water bath. Remove tubes from ice, and immediately immerse in 42 °C water bath for 90 seconds. b. Immediately return both tubes to ice, and let stand on ice for at least 1 additional minute. 13. Place + and tubes in test tube rack at room temperature. 14. Use a 100-1000  µl micropipette with a fresh sterile tip to add 250  µl of sterile LB medium to each tube. Gently tap tubes to mix. This will allow the cells to recover from the heat shock. 15. Use the matrix below as a checklist as + and cells are spread on each plate: 16. Use a 100-1000  µl micropipette with a fresh sterile tip to add 100  µl of cell suspension from the tube onto the LB plate and another 100  µl onto the LB/amp plate. 17. Use a 100-1000  µl micropipette with a fresh sterile tip to add 100  µl of cell suspension from the + tube onto + LB plate and another 100  µl of cell suspension onto + LB/amp plate. [Do not let suspensions sit on plates too long before proceeding to Step 18.] 18. Use sterile glass beads to spread cells over the surface of each plate: a.Obtain four 1.5 ml tubes containing at least five sterilized glass beads. b.Lift lid of one plate, only enough to allow pouring of the beads from one of the 1.5 ml tubes onto the surface of the agar. Replace plate lid; do not set the lid down on the lab bench. Repeat for all plates. c.Use beads to spread bacteria evenly on plates by moving plates side to side several times. Do not move plates in a circular motion. d.Rotate plates  ¼ turn, and repeat spreading motion. Repeat two more times. The object is to separate cells on agar so that each gives rise to a distinct colony of clones. 19. Let plates set for several minutes to allowing the suspension to become absorbed into the agar. Then wrap together with tape. 20. Place plates upside down in 37 °C incubator, and incubate for 12-24 hours, or store at room temperature for approximately 48 hours.5 Results Transformed cells Non-transformed cells LB/amp Bacterial Growth in form of green colonies No growth on plate LB Growth spread across entire plate (bacteria lawn) Growth spread across entire plate (bacteria lawn) Table 1. the E. coli bacterial plates after incubation. Discussion Before the experiment was conducted the results of each plate was hypothesized. It was believed that the plate with only the LB and no plasmids added would grow a lawn of bacteria, this was proven correct by the experiment. The plate with LB and ampicillin but no pGLO was predicted to have no growth, which was also proven correct by the experiment. The plate with LB and ampicillin but the bacteria was administered with the pGLO was predicted to survive the amp but not in very large quantities. Finally, for the plate with only LB but with the pGLO administered to the bacteria it was hypothesized that it would glow, not necessarily in large quantities but at least a little. This was different from the results of the experiment in which the bacteria did not show bioluminescent properties. This can occur for numerous reasons, the lack of bacteria that was transformed, unsterile equipment, improper heat shocking to make the bacteria competent. While all these are the possible reasoning fo r the experiment results the most probable cause for the plates to not grow is the lack of arabinose sugar which is an important part in the expression of the GFPS (see introduction). If the plates lack the arabinose sugar the GFP proteins may not be expressed. This explains why the LB only plate with the pGLO did not produce transformed bacteria. This also draws questions to why the plate with LB and ampicillin and the transformed bacteria. Why would it glow if it didnt have any arabinose sugar? This most likely is explained by the fact that it must have been administered in the LB but not in the others.3 The transformation Efficiency was determined by counting the number of colonies on the LB/amp plate pGFP. Any bacteria that shows light under the UV light must have accepted the plasmids and successfully transformed the desired genes to survive the lb/amp plate and express the GFP gene. Each colony represents one bacteria that has been transformed. Using this the efficiency can be determined. Transformation efficiency is expressed as the number of antibiotic resistant colonies per  µg of pGFP DNA. To find this the mass of the pGFP used must first be determined by the formula Concentration X Volume = Mass. This is shown in figure 1 and was calculated using the formula 0.005  µg / µl x 10  µl = 0.05  µg. Then using the formula to determine the total number of cells per plate the fraction of cells suspended onto the +LB/Amp plate. This is shown in figure 1 and was calculated using the formula .005  µg/510  µl=9.810^-5  µg / µl this number must them be multiplied by 100 because there are approximately 100 cells in use. This is calculated in figure 1 and is solved to be 9.810^-3. To determine the transformants per microgram the formula (total transformed cells/cells per plate)/10000 to find the efficiency in transformants per microgram. This is solved in figure 1 to be 8.673 transformants per microgram. Then the Transformation Efficiency can be found. This is shown in figure 1 which uses the formula (Total cells to start / total microliters) x 100 microliters to find the total number of cells on the plate. Then the formula (Transformants/ Total cells) x100 = percent of efficiency. This is calculated as (8.673 transformants/ 1,960,784,314) x100 to calculate a transformation efficiency of .000004335% or in scientific notation 10x 4.3355 ^ -6 Before conducting the experiment, it was hypothesized that the transformation efficiency would be about 810^-4%. After doing the experiment the transformation efficiency was found to be 4.33510^-6% or 8.673 transformants per microgram. This proves the percentage of efficiency to be significantly lower than hypothesized. The transformation efficiency being lower than expected shows the rarity of this specific form of genetic modification. The experiment tests how rare it is for the genetic modification to occur and demonstrates the results of the modification and its effect on an organism. Citations Griffiths, Anthony JF. Bacterial Transformation. An Introduction to Genetic Analysis. 7th  Edition. U.S. National Library of Medicine, 01 Jan. 1970. Web. 31 Dec. 2016. Bacterial Transformation. SpringerReference (n.d.): n. pag. Cold Spring Harbor  Laboratory. Dolan DNA Learning Center. Web. Reece, Jane B. Campbell Biology, Volume 1. Boston, MA: Peason Learning Solutions, 2011.  Print. Chapter 20 Transfer, Genetics, And Information. BIOTECHNOLOGY: BACTERIAL  TRANSFORMATION* (n.d.): n. pag. Web Lab Center Bacterial Transformation. Lab Center Bacterial Transformation. N.p., n.d.  Web. 03 Jan. 2017. Bacterial Transformation. SpringerReference (n.d.): n. pag. Web. Role As A Student Nurse | Reflection Role As A Student Nurse | Reflection This essay will discuss a significant event in relation to the peoples priorities identified within the Healthcare Quality Strategy. It will also discuss my role as a student nurse in relation to a patient who is receiving palliative care. The essay will demonstrate my understanding and views on reflection and the issues surrounding my practice. I have chosen Gibbs (1988) reflective framework as it has a structured format and six steps which follow in order starting with a description of the event and ending with an action plan for future practice. The steps are Description, Feelings, Evaluation, Analysis, Conclusion and Action Plan (Gray, 2007). Reid, (1993) describes reflection as a process of reviewing an experience of practice in order to describe, analyse, evaluate and so inform learning about practice. In May 2010, the Scottish Government introduced the Healthcare Quality Strategy for NHS Scotland, their aim is to deliver the peoples priorities which are: firstly caring and compassionate staff and services, good communication and a clear explanation about conditions and treatments, effective teamwork between clinicians, patients and others, a clean and safe care environment, continuity of care and clinical excellence. Secondly, to bring together the patients priorities as well as the values of everyone working within the NHS Scotland. Thirdly, by applying these three interventions and putting them into practice improvements within priority areas of the Healthcare Quality Strategy will be met (Scottish Government, 2010, p.6). This reflective account will identify the way in which these priorities were met in relation to my significant event and it will also confirm their importance. Description Whilst on my Management Placement I met Janet, she is 65 years old and has been admitted to my ward for palliative care. She has end stage bowel cancer and is awaiting a bed at the local hospice. All names have been changed to protect her identity and maintain confidentiality (NMC, 2008). Janets GP has requested that she be admitted onto the ward as she is now confined to bed because of increased pain and a general deterioration of her condition (Dougan and Colquhoun, 2006). Feelings Whilst carrying out my training as a student nurse, I have met a number of palliative patients and you feel helpless as to how to care for these patients. With regard to this experience my feelings are sadness, anger and helplessness. In relation to the peoples priorities, I must provide the best care for my patient and be caring and compassionate always (Scottish Government, 2010, p.6). The level of involvement from all members of the multidisciplinary team, the care and compassion shown to this patient at all times was very humbling (NMC, 2008). I was privileged to have the opportunity to be involved in Janets care as well as working within a ward of clinical excellence (Scottish Government, 2010, p.6). Evaluation A holistic approach with continuity of care is extremely important and all nurses have a central role in providing information, care and support (Kennedy and Lockhart, 2007). Excellent communication and good interpersonal skills are essential in order to provide Janet with the care she requires. Also when dealing with members of the multi-disciplinary team, effective teamwork is imperative again providing Janet and her family with continuity of care and the clinical excellence they require from all staff. I feel the Quality Strategy aim was achieved, as all staff when looking after Janet dealt with her in a caring and reassuring manner at all times, in turn providing Janet with the best care, whilst she was on the ward (Scottish Government, 2010). Analysis Palliative care helps to improve the quality of life for patients and their families who have to come to terms with the difficulties associated with life threatening illness. It is about identifying the patients needs through accurate assessment, good symptom control and sensitive nursing care (Scottish Partnership for Palliative Care, 2012). Over the next few days whilst looking after Janet and taking part in the ward rounds, I was able to plan Janets care. Whilst preparing Janets care plan I put some goals into place with the aim of making Janets stay in hospital as comfortable as possible and also to determine if Janets physical condition was improving or deteriorating (Richards and Edwards, 2008). When planning Janets care I took a holistic approach and looked at her physical, psychological, social, emotional and environmental needs. This included not only the patient, the hospital and any multi-disciplinary team members but also her husband and any other family members she would like involved (Dougherty and Lister, 2011). As Janets condition could change from day to day her care plan was reviewed on a daily basis and updated accordingly (Dougan and Colquhoun, 2006). As Janet was bed bound she required the assistance of two members of staff to assist her with her Activities of Daily Living (Hamilton and Price, 2007). Good personal care from the nurses and the nursing auxiliaries is paramount. As Janet is lying in the same position for a long period of time, the development of pressure ulcers can become a concern as her skin can breakdown quickly. The use of the Waterlow Risk Assessment Tool was used in assessing and establishing the needs of the patient; this involved regular positional changes, a specially prepared mattress for her bed or a cushion for their chair (Waterlow 2012). Following assessment the nurse developed a care plan in order to help maintain patient skin integrity and any actions or implementations which needed to be carried out (Anderson and Fletcher, 2007). In addition to caring for Janet on the ward I was given the opportunity to go to the Hospice and meet with Janets palliative care nurse and also be included in her multi-disciplinary team meeting. This meant I learnt more about Janets condition and therefore provided her with the best care I could whilst she is on the ward. Palliative care patients must have good symptom control of their pain and also nausea and vomiting. Janet was placed on a syringe driver to control her pain. A syringe driver is a small portable pump which is attached to the skin by a cannula and gives the patient a continuous dose of analgesics and/or anti-emetics (MacMillan Cancer Support, 2012). When dealing with patients who are receiving palliative care the peoples priorities are relevant. All nurses must be able to make the patient as comfortable as possible, be aware of their wants and needs and provide a safe and clean environment (Scottish Government, 2010, p.6). On reflection this event has brought further awareness of the level of involvement of staff members from nursing auxiliaries, nurses and consultants. This was shown by the number of multi-disciplinary meetings, assessments, re-assessments, written correspondence and working hours placed into looking after this patient. All staff members were committed to providing the correct input and treatment for this patient and to make her last few days as comfortable as possible (Scottish Government, 2008). Conclusion This reflection has demonstrated the difficultly in caring for palliative care patients. It requires a holistic approach to ensure that the patient and their family receive the best possible attention. Nurses have a duty to ensure that the care they deliver is of an acceptable standard (NMC, 2008). While participating in ward rounds I took on board the doctors instructions and carried out and prioritised the care of my patient, therefore making Janets stay in hospital as comfortable as possible. The code provides values, which can be adapted to any setting and as long as these are followed, nurses will be able to carry out their legal and professional duty of care (NMC, 2008). Identify Steps All staff on this ward were experienced in dealing with palliative care patients. However as a student you can be hesitant when communicating with patients and families at this difficult time for fear of saying something incorrect. Therefore education and training not only for students but for all staff across all care settings should be paramount in order to provide good palliative and end of life care (Scottish Partnership for Palliative Care, 2012). In relation to my transition from student to nurse, palliative care does not only deal with cancer patients but with patients who have long term chronic illnesses such as multiple sclerosis, heart disease and respiratory problems, therefore palliative care is relevant today and also in the future because we will be caring for an ageing population (Scottish Partnership for Palliative Care, 2012) In conclusion and in relation to The Healthcare Quality Strategy I feel that Janet was looked after in a caring and compassionate manner at all times and the nursing auxiliaries and nurses looking after Janet worked well together putting her first and treating her with respect at all times (Scottish Government, 2010). Action Plan Palliative care is a sensitive subject to deal with but I feel that as long as you treat the patient as an individual, listen to their wants and needs, then a positive outcome can be accomplished. Communication and good interpersonal skills are once again highlighted as an essential part of good nursing practice. I must be an effective communicator and be able to provide a high standard of care at all times and this is imperative when dealing with palliative care patients. I must keep my knowledge and skills up to date (NMC, 2008). Whilst on this placement I was expected to take control of situations and be able to communicate and deal with whatever is presented to me. You have to be able to stand on your own two feet and this will be imperative in my transition to becoming a registered nurse. Conclusion In conclusion and in relation to The Health Quality Strategy and my transition from student to nurse, Bowie (2010) states that positive practice should be highlighted as it allows others to learn from it and provides opportunities to improve the safety of patient care. Reflecting on these events has helped me to identify areas where practice needs improvement and given me a greater insight into my own role as a registered nurse.