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Transformation Christopher & Miniprep
Objective: The purpose of this lab is to use the chemically competent cells that we created to learn how to transform bacteria using various plasmids. The transformed cells will then be harvested and grown for DNA extraction and purification using miniprep. One plasmid will be plated using different concentrations to test the efficiency of the competent cellsAlso we will test the cells for contamination of mold or naturally ampicillin resistant bacteria. Lab Protocol: Plasmid Transformation and Miniprep The three competent cell 50 L aliquots were first taken out of an -80 C freezer and thawed on ice. The cell tubes were then labeled by the name of the plasmid that would be used in the transformation reaction. The plasmids were as follows: mCherry, tetR, and GFP. Three aliquots of sterile SOC media were taken out of a freezer and placed into a 37 C incubator. Five LB-amp plates were also taken out of the freezer and placed into the incubator. Once the competent cells thawed 50 ng of each plasmid was micropipetted into its own tube of cells. The concentration of the three plasmids and the subsequent volumes used were as follows: tetR mCherry GFP 85.2ng/ L , 0.587 L
147.06ng/ L , 0.340 L 85.2ng/ L , 0.587 L
The tubes were then incubated on ice for 30 minutes. After incubation the cells were heat shocked in a 42 C water bath and immediately placed back on ice for two minutes. 950 L of room temperature SOC media were transferred into each tube. The tubes were then shaken horizontally in a 37 C incubator for one hour. After the incubation the cells were then spread plated on the LB-amp plates that had been incubating. This process began with putting five sterile colirollers onto each plate. 200 L of both the mCherry transformed bacteria and the tetR transformed bacteria were then micropipetted onto their own plates and were spread on the plate by swirling
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the colirollers around the plates until the bacteria had been spread evenly. For the GFP transformed bacteria, three different concentrations of the bacteria were prepared and then plated using the same technique. The first two had 10 L and 100 L of original plasmid bacteria and 190 L and 100 L of LB media to keep the final volume 200 L. The third began with 800 L that was centrifuged at 3000 rpm for one minute. The supernatant was disposed of and the pellet was resuspended in 200 L of LB media. After the bacteria was able to soak into the plates for ~3 minutes the colirollers were removed and the plates were then incubated for 18 hours in an inverted position. After the allotted time the plates were taken out of the incubator. The three plates with GFP were examined. The colonies on each plate were counted and the transformation efficiency was then calculated. Three colonies on each of the other two plates, tetR and mCherry, were then singled out and circled with sharpie on the bottom of the plate. Six sterile culture tubes were then obtained and filled with 5mL of LB-amp media. Each colony was then sampled using a sterile applicator. The colonies were each mixed into their own culture tube by swirling the applicator in the LB-amp. All tubes were then labeled with their plasmid type and colony number. These six tubes were then incubated in the 37 C shaking incubator overnight at 225 rpm. The plates of tetR and mCherry were wrapped in parafilm and stored in the 4 C refrigerator and the other plates were discarded. The following day the culture tubes were taken out of the incubator and the DNA was extracted using the Sigma GenElute Plasmid Miniprep kit. First the cells were harvested by transferring 1.5mL of each colony into a sterile microcentrifuge tube. The cells were then pelleted using a centrifuge for one minute at 13,000 rpm (full speed). The supernatant was poured off into a waste container and the pellet was resuspended in 200 L of the Resuspension Solution. The cells were then lysed, by adding 200 L of the Lysis Solution. The contents of each tube was then mixed by inverting the tubes 10X. The Lysis Solution was then neutralized, by adding 350 L of the Neutralizing Solution. Again the contents were mixed using the inversion technique above. The cells were then centrifuged for 10 minutes at full speed. During this centrifugation, six GenElute Miniprep binding columns were inserted into six collection tubes. 500 L of the Column Preparation Solution were then added to the collection tubes. This apparatus was then
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centrifuged at full speed for a minute, and the flow-through was poured out. The cleared lysates that were created by centrifuging the lysed cells were then poured into their own collection/column tube. This was then centrifuged at full speed another for minute, and the flow-through was discarded. The columns were then washed, by adding 500 L of the Optional Wash Solution to each tube. Again the tubes were centrifuged at full speed for one minute. The flowthrough was discarded. Next the tubes were washed again using the same procedure as stated above with 750 L of the Wash Solution. Another centrifuging after the above wash was discarded removed any remaining ethanol in the column. Finally, the DNA was collected. This was done,by adding 100 L of sterile dH2O to the column, and centrifuging one final time at full speed for one minute. The samples of DNA were then tested using a nanodrop spectrophotometer. This measured the concentrations of each sample of DNA. Control Experiments First one vial of the competent cells and two plates were collected. One plate contained LB-amp and one contained LB. The cells were thawed out on ice and then streak plated. 10 L of the cells were micropipetted onto each plate. A sterile Q-tip was then used to spread the bacterial cells. The two plates were then transferred into the 37 C shaking incubator for growing overnight. The following day the plates were taken out of the incubator and observed. Results: The original volumes and colonies counted for the GFP plates were as follows.
10L 0 colonies 100L 3 colonies 800L 37 colonies
The concentrations of the six DNA samples were as follows:
tetR #1 9.04 tetR #2 7.28 tetR#3 7.76 mCherry #1 15.36 mCherry #2 16.02 mCherry #3 16.71
The control LB plate showed confluent cell growth. This was consistent with the expected results. Also on the LB-amp plate there was no growth, which was also expected.
Christopher Wilkerson Interpretation:
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The concentrations of each of the DNA samples seem very low, however this could be due to an old miniprep kit. The age of the miniprep kit could have led to chemical degradation. This would have affected the ability of the kit to extract the DNA. The control plates yielded results that showed that the competent cells had no mold and were not damaged or contaminated during their preparation. Also the LB-amp plates were working and no cells were naturally resistant. Post-Lab questions 1. What are the Biobrick parts that you used in your transformation reactions? tetR, GFP, and mCherry 2. Why do you need a promoter upstream to your gene for proper gene expression? Without the promoter, the transcription complex would not form and no protein would ultimately be produced. 3. From the Biobrick website, how will you insert the promoter upstream to your gene? Restriction enzymes will be used to insert the plasmid upstream of the gene. 4. Why are plates incubated at 37 C upside down? And why are transformed bacteria only incubated for 16-18h after plating? What are satellite colonies? The temperature is most conducive to growing bacteria and the inversion prevents water droplets from condensation from falling and crushing any colonies. If the bacteria are allowed to incubate any longer than18 hrs, they will cease to be viable. Satellite colonies are colonies that benefit from the production of proteins that neutralize the effects of the ampicillin and are able to undergo mitosis. 5. How do we sterilize our solutions and media before we use it for bacterial or DNA work? How do you know if a solution or an object is sterile? We autoclave solutions and media to sterilize them. Any autoclaved solution or media will have tape with black stripes on it. 6. What was the transformation efficiency of your competent cells? Please show your calculations. For calculating amount of plasmid plated:
o
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10 L -
800 L For calculating transformation efficiency:
The average of the two efficiencies is the overall efficiency 762.31transformants/ng plasmid. 7. Please interpret the results from your control experiments. Did anything weird grow on your plates? The control plates came out as expected. Nothing grew on the LB-amp plate and confluent cell growth was observed on the LB plate. This proved that the cells weren't damaged or contaminated during their preparation and that the plates were good. 8. What was one cool thing you learned from the lab and what is one mistake you learned in lab this week? The process of transformation is one very cool thing that I learned how to do in the lab. Also I learned not to thaw cells without ice. This seemed very counterintuitive. RFP red fluorescent protein This protein came from cloning proteins in the Discosomastriatacoral. Scientists discovered this protein because they were searching for a substitute for green fluorescent protein. The excitation spectrum has a major peak at 558 nm while the emission spectrum wavelength peak is at 583 nm.1
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http://www.microscopyu.com/articles/livecellimaging/fpintro.html