Optimizing the Rate of Bacterial Transformation
Purpose
The purpose of this project is to see what types of natural antibiotics can effect bacterial transformation in a positive way, that is to say, what natural antibiotics can transform E. coli.
Significance
Using bacteria to create insulin for diabetes patients is a new and effective way of helping people who suffer from this disease. This project will utilize the pGreen plasmid as a way of observing transformation rates of E. coli with different types of natural antibiotics.
Hypothesis
H1: Ampicillin will transform E. coli the fastest
Procedures
Chemical Safety:
1) During the part of the experiment that will be performed at the school, all chemicals (Nutrient Agar) will be used in accordance with the MSDS.
2) The use of all chemicals will be done while wearing gloves, aprons, and goggles.
3) All chemicals that release fumes will be placed under the fume hood to prevent the release of fumes into the classroom.
4) A fire blanket, fume-hood, fire extinguisher, eyewash station, and shower are available for use.
5) Upon leaving the lab, hands will be washed and all chemicals will be stored in a locked ventilated chemical storeroom.
Chemicals and Heat:
1) Goggles and aprons will be worn.
2) During heating, potholders (thermal gloves) will be used to handle hot surfaces and equipment coming off the hot plate.
3) No containers will be sealed when heated. In the event a closed container needs to be heated, a pneumatic trough or pressure release valve will be used.
Electrical Power Safety
1) Limit the use of high power devices around water.
2) Never used frayed or cut wires when plugging in wires.
3) Never overload the outlet and use a surge protector.
4) All electrical devices should have an emergency cut off switch or “kill” switch.
5) Shut off all electrical devices when not in use or when you leave the room.
Sharp Objects
1) Use caution when using scalpel.
2) Restrict the use of sharp objects to a minimum.
3) Do not use broken “sharps”.
4) Cover or cap scalpel after each use.
5) Discard used syringes, needles, or scalpels into an approved sharps container.
6) Bring all needles in a sharps container to a local fire station for disposal.
BSL 1 Aseptic Techniques
Aseptic Techniques
1) Upon entering the lab, wash hands all the way to the elbows with antibacterial soap.
2) Before and during experimentation wear rubber gloves, apron, and goggles.
3) All glass and other materials should be autoclaved for 30minutes at 20 PSI, 120 degrees Celsius.
4) Any open flask, container, or beaker should be covered with aluminum foil when placed in the autoclave.
5) Use 10% bleach solution and wipe down the lab area and tabletop.
6) Transfer of any culture will be done with a mechanical pipette.
7) Place a biohazard sign in plain view for all to see.
8) Use sterile loops and needles that don’t need to be flamed.
9) The top of the culture-tube will be flame heated before inserting a loop or needle for culture transfer.
Autoclave
1) Always wear goggles and aprons when using the autoclave.
2) The bottom of the autoclave should have about two inches of clean distilled water before operation.
3) Check the pressure release valve prior to its use (it should be clear)
4) Use potholders when opening or handling hot surfaces
5) Opening the autoclave can be done only after it has cooled (gauge should read zero) and all the steam has been allowed to escape.
6) Place the control knob in the straight up position. This allows the unit to operate at 16-21 PSI range.
Culturing
1) On the underside of each Petri dish plate, label with a wax pencil.
2) Heat nutrient agar in a microwave or water bath to 50 degrees C
3) Pour a thin layer of liquefied agar onto the bottom of each plate, cover immediately.
4) Let agar plate solidify. It will solidify at about 42 degrees C
5) After the agar has solidified, streak plate using the proper technique.
6) Once the plates have been streaked, invert and close the Petri dish. Tape the edges and incubate them at 25 degrees C for 24-48 hours.
Streaking Plates
1) Sterilize work area and safety equipment.
2) Take out plates from fridge and allow them to cool to room temp.
3) Take out bacteria to be streaked on plates.
4) Take a sterile inoculating loop and scrape off growth from bacterial culture.
5) Place inoculating loop into sterile broth of distilled water and agitate.
6) Dip sterile swab into wash.
7) Lift up lid of plate and streak wet swab onto plate being sure to cover the entire area. Plate can be streaked multiple times in a sweeping motion to ensure total coverage.
8) When done streaking, place in incubator upside-down.
9) Dispose of materials.
Cleaning up
1) After everyday of experimentation, clean the lab area with 10% bleach.
2) Wash hands with 10% bleach
3) Clean with 10% bleach all glassware and related items. Autoclave new equipment as needed
4) Petri dishes and other disposable equipment will be placed in a plastic bag and autoclaved for 30 min at 20 PSI, 120 degrees C and disposed of.
5) All related glassware and other items will be autoclaved for 30 min at 20 PSI, 120 degrees C.
Experimental Setup
Making Agar
1) Loosen lid on Agar Bottle
2) Heat to boiling in microwave (5-7 min)
3) Swirl after heating up
4) Repeat until agar is completely dissolved
5) Pour 16 plates
6) Add 4ml of 1% Ampicillin to agar when cool enough to hold
7) Pour 16 plates
8) Make 1% dilution of: honey, garlic, and cinnamon in 4ml of sterile water
9) Heat up 2 more bottles of agar and separate into four separate beakers
10) Add each solution to one of the beakers and label
11) Pour 16 plates for each solution
Preparing pGreen Plasmid
1) Transfer 250 µl of ice-cold calcium chloride into +pGreen microtube
2) Place on crushed ice
3) Using a sterile loop, pick up bacterial colonies for slant and immerse in solution and spin around
4) Repeatedly pipette in and out to suspend bacteria
5) Place back on ice
6) Using a sterile inoculating loop, place in plasmid solution and transfer into +pGreen microtube and swirl
7) Put back on ice for 15 min
8) Heat shock at 42*C in a water bath for exactly 90 sec
9) Put back on ice for 1 min
10) Add 250 µl LB Luria Broth
11) Place in test tube rack and allow recovery for 5- 15 minutes
12) Pipette 100 µl onto each plate
· Pour 4-6 glass beads onto each plate
· Use a back-and-forth motion to spread cells
· Let rest for several minutes
· Remove by opening plate over a container
13) Wrap plates and label
14) Incubate over night at 37*C for 24-36 hours
Data Collection
1) Take out of incubator and observe whether each plate glows
2) Record number and type that glow in regular light
3) Place under UV light and record number and type that glow
4) Organize data into chart
Results
X = No Glow | Ampicillin | Honey | Garlic | Cinnamon |
1 | X | X | X | X |
2 | X | X | X | X |
3 | X | X | X | X |
4 | X | X | X | X |
5 | X | X | X | X |
6 | X | X | X | X |
7 | X | X | X | X |
8 | X | X | X | X |
9 | X | X | X | X |
10 | X | X | X | X |
11 | X | X | X | X |
12 | X | X | X | X |
13 | X | X | X | X |
Conclusion
In conclusion, the hypothesis was rejected. All of the plates that used a natural antibiotic failed in transforming the bacteria. The plates containing ampicillin in the agar also did not transform the bacteria.
Bibliography
"Biochemistry : Genetic Transformation(using Bacteria and the PGLO Plasmid)." ZAMP
BioWorld. Web. 9 Sept. 2010.
<http://www.zampbioworld.org/material/bioch_lab/bc043_a.php>.
"Gene Regulation and the Arabinose Operon." Yahoo! Search - Web Search. Web. 12
Oct. 2010. <http://74.6.238.254/search/srpcache?ei=UTF-8&p=arabinose
operon&fr=yfp-t-701&rs=0&u=http://cc.bingj.com/cache.aspx?q=arabinose
operon&d=4593918629643020&mkt=en-US&setlang=en-US&w
=48326f91,5cd77069&icp=1&.intl=us&sig=96PBlxmixgHsg0tR.Z1d8g-->.
"L-Arabinose(CAS:5328-37-0)::CHEM-ONLINE.org." ::CHEM-ONLINE.org::
Extensive Chemical Information Online Serve Site. Web. 27 Aug. 2010.
<http://www.chem-online.org/food-ingredient/arabinose.htm>.
"Nature Protocols: Fast and Efficient Genetic Transformation of Sugar Beet by
Agrobacterium Rhizogenes." Featured Protocols : Nature Protocols. Web. 11
Oct. 2010. <http://www.natureprotocols.com/2010/05/11/fast_and_efficient
_genetic_tra.php>.
"Society for Science & the Public - Intel ISEF - Home Page." Society for Science & the
Public - Home. Web. 05 Nov. 2010. <http://www.societyforscience.org/isef/>.
