Team:WarrenCentral WCC IN

From 2012hs.igem.org

We are Warren Central High School from Indianapolis, Indiana. We are made up of students who take Scientific Innovations, the fourth and final class in the Project Lead the Way curriculum. This class is an independent study course, and we chose to focus on the iGEM competition.


Exposure to mercury is a widespread problem that affects many people all over the world. Most people ingest mercury through water sources. Mercury in water can arise from runoff from farms, chemical and industrial plants, household products in the trash, and sewage. Three types of mercury can adversely affect humans. Elemental, inorganic, and methyl mercury can all harm humans if ingested. Inorganic mercury is the most common form in drinking water and can cause kidney damage if enough is taken in. Methyl mercury is found in fish and humans can be exposed if they eat too much mercury-containing fish. Mercury ingestion can cause both acute and chronic symptoms.

We are using Saccharomyces cerevisiae yeast as a tool to detect mercury. In yeast, there are a number of transcription factors and genes that respond to oxidative stress and toxic metals. The yes associated protein (YAP) family is a family of transcription factors that is involved with oxidative stress regulation and redox homeostasis. They affect a number of genes, but we are focusing on GSH1 and GSH2. These genes are involved in the glutathione pathway. Glutathione is an antioxidant that protects the cell from oxidative stress.

In order to detect mercury, we are using several biological parts included in the BioBrick. The Kozak + mCherry translational unit is being used to give off a red fluorescent glow when the mercury is detected. In the plasmid, we will include the GSH2 promoter and the ADH1 terminator.

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Contents

Team

-Charlie Dant

Charlie Dant is attending Depaw University in the fall. He is very active as second Vice President of our student council, and he plays on our schools varsity basketball and baseball team. He will be playing for Depaw next year. Charlie has also been voted Warren Central's Mr. Basketball and had a great time on prom court.


- Samantha Deitz

Sam is a biology graduate student at IUPUI. She currently works in a research lab and a teaching program called K-12. She enjoys her time here at Warren Central teaching the biomed students and assisting with labs.Sam is a very essential part of the team, not only because she is smart, but because she is fun and beautiful.


-Cheyenne Harvey

Cheyenne is a senior and a dedicated member of Student Council. When she isn't doing volunteer work, she spends most of her time taking care of her beautiful puppy, Tink. Cheyenne plans on attending the University of Indianapolis in the fall where she will be majoring in radiology. In the event that her career as a professional rapper falls through, Cheyenne hopes to instead become a radiologist, specializing in ultrasounds.


-Rebekah Hodge

Bekah is currently a captain of the Warrren Central Speech team and member of Student Council, Latin club, National Junior Classical League, and Indiana Junior Classical League. She also works part time as a waitress for MCL Restaurant & Bakery. She is planning on attending Indiana University – Bloomington and majoring in Biology/PreMed. Eventually, she hopes to become a pediatrician and run her own family practice.


-Sarah Huh

Sarah Huh? will be attending IU Bloomington in the fall. She is going to major in chemistry and go into pre-med to become a pediatrician. Currently Sarah is the 1st vice president of student council, in National Honor Society, Just Say No,and Fellowship of Christian Athletes. She was also the captain of her high school soccer team. Sarah plays travel soccer, sings for church, and paints soccer fields.


-Diamond Jones

Diamond is planning on majoring in genetics at Purdue University. She is actively involved in her school’s honors band and National Honors Society. Diamond has put in countless hours of volunteer work at the Indianapolis Humane Society, churches, retirement homes, and local food pantries. Her future plans include becoming a genetic counselor, and creating a gene that allows her to live for eternity.


-AJ McAdams

AJ teaches the PTLW Biomedical courses at Warren Central. Mr.McAdams also coaches the boys soccer team at school. Other than teaching, he enjoys his time with his family, playing sports, and talking about how cute his sons are.


-Myka Norris

Myka is planning on majoring in nursing at IUPUI. Most of her time is taken up by her job at Steak n Shake. However, in her free time she enjoys running and hanging out with family and friends.


-Sarah Perun

Sarah will be studying biology and following the pre-med track at Purdue University. She enjoys traveling, eating, sports, music, reading, and shopping. Sarah is involved with the varsity golf team, band, and National Honors Society. Boiler up!


-Patrick Steuer

The year was 1994, it was a warm spring evening in the month of April, the 11th if I recall correctly, when Patrick Steuer came screaming into the world, changing it in a way it would never truly recover from. Patrick doesn’t do a whole lot aside from chilling, but he does it extremely well. He is going to IU and plans on majoring in human biology. His goal is to eventually become a genetic counselor.


-Amanda Weber

Amanda is attending University of Southern Indiana in the fall. Amanda enjoys hanging out with friends, working at the gym, and practicing her gymnist skills.


-Brandon Whatley

Brandon Whatley is a top-notch student at Warren Central. He is an active member in student council, the reigning president of the Spanish Club, a member of Connection Show Choir, a member of the Fun Monkey Improv team, and a golf player. He plans on attending Indiana University in Bloomington in the Fall of 2012 and majoring in biology. He also enjoys long walks on the beach and the feel of fingers running through his wavy hair. His number is 317-345-6864. Call or text him any time. ;)


-Lewis Woods

Lewis Manning Woods IV. is a senior and plans on attending IU Bloomington in the fall. He has not yet declared his major but wants to go to medical school, be a doctor and have his own practice when he gets out of school. Outside of school he is very active in his church, and captained the High School soccer team. He also plays tennis for recreation.

Project

From partsregistry.org

[[Protocols/Competent Cells]]


Overview


This protocol is a variant of the Hanahan protocol [1] using CCMB80 buffer for DH10B, TOP10 and MachI strains. It builds on Example 2 of the Bloom05 patent as well. This protocol has been tested on NEB10, TOP10, MachI and BL21(DE3) cells. See OWW Bacterial Transformation page for a more general discussion of other techniques. The Jesse '464 patent describes using this buffer for DH5α cells. The Bloom04 patent describes the use of essentially the same protocol for the Invitrogen Mach 1 cells. This is the chemical transformation protocol used by Tom Knight and the Registry of Standard Biological Parts.


Materials


• Detergent-free, sterile glassware and plasticware (see procedure)

• Table-top OD600nm spectrophotometer

• SOB CCMB80 buffer

• 10 mM KOAc pH 7.0 (10 ml of a 1M stock/L)

• 80 mM CaCl2.2H2O (11.8 g/L)

• 20 mM MnCl2.4H2O (4.0 g/L)

• 10 mM MgCl2.6H2O (2.0 g/L)

• 10% glycerol (100 ml/L)

• adjust pH DOWN to 6.4 with 0.1N HCl if necessary

o adjusting pH up will precipitate manganese dioxide from Mn containing solutions.

• sterile filter and store at 4°C

• slight dark precipitate appears not to affect its function


Procedure

Preparing glassware and media


Eliminating detergent


Detergent is a major inhibitor of competent cell growth and transformation. Glass and plastic must be detergent free for these protocols. The easiest way to do this is to avoid washing glassware, and simply rinse it out. Autoclaving glassware filled 3/4 with DI water is an effective way to remove most detergent residue. Media and buffers should be prepared in detergent free glassware and cultures grown up in detergent free glassware. Prechill plasticware and glassware Prechill 250mL centrifuge tubes and screw cap tubes before use.


Preparing seed stocks


• Streak TOP10 cells on an SOB plate and grow for single colonies at 23°C

o room temperature works well

• Pick single colonies into 2 ml of SOB medium and shake overnight at 23°C

o room temperature works well

• Add glycerol to 15%

• Aliquot 1 ml samples to Nunc cryotubes

• Place tubes into a zip lock bag, immerse bag into a dry ice/ethanol bath for 5 minutes

o This step may not be necessary

• Place in -80°C freezer indefinitely.


Preparing competent cells


• Ethanol treat all working areas for sterility.

• Inoculate 250 ml of SOB medium with 1 ml vial of seed stock and grow at 20°C to an OD600nm of 0.3. Use the "cell culture" function on the Nanodrop to determine OD value. OD value = 600nm Abs reading x 10

o This takes approximately 16 hours.

o Controlling the temperature makes this a more reproducible process, but is not essential.

o Room temperature will work. You can adjust this temperature somewhat to fit your schedule

o Aim for lower, not higher OD if you can't hit this mark

• Fill an ice bucket halfway with ice. Use the ice to pre-chill as many flat bottom centrifuge bottles as needed.

• Transfer the culture to the flat bottom centrifuge tubes. Weigh and balance the tubes using a scale

o Try to get the weights as close as possible, within 1 gram.

• Centrifuge at 3000g at 4°C for 10 minutes in a flat bottom centrifuge bottle.

o Flat bottom centrifuge tubes make the fragile cells much easier to resuspend

• Decant supernatant into waste receptacle, bleach before pouring down the drain . • Gently resuspend in 80 ml of ice cold CCMB80 buffer

o Pro tip: add 40ml first to resuspend the cells. When cells are in suspension, add another 40ml CCMB80 buffer for a total of 80ml

o Pipet buffer against the wall of the centrifuge bottle to resuspend cells. Do not pipet directly into cell pellet! o After pipetting, there will still be some residual cells stuck to the bottom. Swirl the bottles gently to resuspend these remaining cells

• Incubate on ice for 20 minutes

• Centrifuge again at 3000G at 4°C. Decant supernatant into waste receptacle, and bleach before pouring down the drain.

• Resuspend cell pellet in 10 ml of ice cold CCMB80 buffer.

o If using multiple flat bottom centrifuge bottles, combine the cells post-resuspension

• Use Nanodrop to measure OD of a mixture of 200 μl SOC and 50 μl of the resuspended cells

o Use a mixture of 200 μl SOC and 50 μl CCMB80 buffer as the blank

• Add chilled CCMB80 to yield a final OD of 1.0-1.5 in this test.

• Incubate on ice for 20 minutes. Prepare for aliquoting

o Make labels for aliquots. Use these to label storage microcentrifuge tubes/microtiter plates

o Prepare dry ice in a separate ice bucket. Pre-chill tubes/plates on dry ice.

• Aliquot into chilled 2ml microcentrifuge tubes or 50 μl into chilled microtiter plates

• Store at -80°C indefinitely.

o Flash freezing does not appear to be necessary

• Test competence (see below)

• Thawing and refreezing partially used cell aliquots dramatically reduces transformation efficiency by about 3x the first time, and about 6x total after several freeze/thaw cycles. Measurement of competence

• Transform 50 μl of cells with 1 μl of standard pUC19 plasmid (Invitrogen)

o This is at 10 pg/μl or 10-5 μg/μl

o This can be made by diluting 1 μl of NEB pUC19 plasmid (1 μg/μl, NEB part number N3401S) into 100 ml of TE

• Incubate on ice 0.5 hours. Pre-heat water bath now.

• Heat shock 60 sec at 42C

• Add 250 μl SOC

• Incubate at 37 C for 1 hour in 2 ml centrifuge tubes, using a mini-rotator

o Using flat-bottomed 2ml centrifuge tubes for transformation and regrowth works well because the small volumes flow well when rotated, increasing aeration.

o For our plasmids (pSB1AC3, pSB1AT3) which are chloramphenicol and tetracycline resistant, we find growing for 2 hours yields many more colonies

o Ampicillin and kanamycin appear to do fine with 1 hour growth

• Add 4-5 sterile 3.5mm glass beads to each agar plate, then add 20 μl of transformation

o After adding transformation, gently move plates from side to side to re-distribute beads. When most of transformation has been absorbed, shake plate harder

o Use 3 plates per vial tested

• Incubate plates agar-side up at 37 C for 12-16 hours

• Count colonies on light field the next day

o Good cells should yield around 100 - 400 colonies

o Transformation efficiency is (dilution factor=15) x colony count x 105/µgDNA

o We expect that the transformation efficiency should be between 1.5x108 and 6x108 cfu/µgDNA 5x Ligation Adjustment Buffer

• Intended to be mixed with ligation reactions to adjust buffer composition to be near the CCMB80 buffer

• KOAc 40 mM (40 ml/liter of 1 M KOAc solution, pH 7.0)

• CaCl2 400 mM (200 ml/l of a 2 M solution)

• MnCl2 100 mM (100 ml/l of a 1 M solution)

• Glycerol 46.8% (468 ml/liter)

• pH adjustment with 2.3% of a 10% acetic acid solution (12.8ml/liter)

o Previous protocol indicated amount of acetic acid added should be 23 ml/liter but that amount was found to be 2X too much per tests on 1.23.07 --Meagan 15:50, 25 January 2007 (EST)

• water to 1 liter

• autoclave or sterile filter

• Test pH adjustment by mixing 4 parts ligation buffer + 1 part 5x ligation adjustment buffer and checking pH to be 6.3 - 6.5

• Reshma 10:49, 11 February 2008 (CST): Use of the ligation adjustment buffer is optional.




[[Protocols/Transformation]]

Transforming Competent Cells


Estimated time: 3 hours (plus 12-14 hour incubation)

It is important to note that we have tested transformations of the distribution kit with this protocol. We have found that it is the best protocol to use with BioBrick parts and ensures the highest efficiency for the transformation. This protocol may be particularly useful if you are finding that your transformations are not working, or yielding few colonies.

Materials

•Resuspended DNA (Resuspend well in 10ul dH20, pipette up and down several times, let sit for a few minutes)

• Competent cells (50ul per transformation)

• Ice

• 42º water bath

• 37º incubator

• SOC (check for contamination!)

• Petri dishes with LB agar and appropriate antibiotic (two per transformation)


Protocol


1. Start thawing the competent cells on crushed ice.

2. Add 50 µL of thawed competent cells and then 1 - 2 µL of the resuspended DNA to the labelled tubes. Make sure to keep the competent cells on ice.

3. Incubate the cells on ice for 30 minutes.

4. Heat shock the cells by immersion in a pre-heated water bath at 42ºC for 60 seconds. A water bath improves heat transfer to the cells.

5. Incubate the cells on ice for 5 minutes.

6. Add 200 μl of SOC broth (make sure that the broth does not contain antibiotics and is not contaminated)

7. Incubate the cells at 37ºC for 2 hours while the tubes are rotating or shaking. Important: 2 hour recovery time helps in transformation efficiency, especially for plasmids with antibiotic resistance other than ampicillin.

8. Label two petri dishes with LB agar and the appropriate antibiotic(s) with the part number, plasmid, and antibiotic resistance. Plate 20 µl and 200 µl of the transformation onto the dishes, and spread. This helps ensure that you will be able to pick out a single colony.

9. Incubate the plate at 37ºC for 12-14 hours, making sure the agar side of the plate is up. If incubated for too long the antibiotics start to break down and un-transformed cells will begin to grow. This is especially true for ampicillin - because the resistance enzyme is excreted by the bacteria, and inactivate the antibiotic outside of the bacteria.


[[Protocols/Ligation]]



After following our restriction digest protocol (which uses 500ng of DNA) you may follow these steps for ligation.


1. Add 11ul of dH20

2. Add 2ul from each sample you will be ligating (destination plasmid, and part at roughly 10ng/ul)

3. Add 2ul of T4 DNA Ligase Reaction Buffer

4. Add 1ul of T4 DNA Ligase

5. Mix well, and spin down.

6. Incubate for 30min at 16C and 20min at 80C to heat kill.

7. Use 2ul of ligation to transform into competent cells.


[[Protocols/Restriction Digest]]


At iGEM HQ we use this protocol for restriction digests along with enzymes purchased from NEB.


Materials


• PCR tube

• dH20

• Enzymes (EcoRI, XbaI, SpeI, PstI)

• BSA

• Enzyme Buffer (NEBuffer 2)*

Notes: You should keep all materials on ice.


Protocol

1. Add 500ng of DNA to be digested, and adjust with dH20 for a total volume of 42.5ul.

2. Add 5ul of NEBuffer 2 to the tube.

3. Add 0.5ul of BSA to the tube.

4. Add 1ul of your first enzyme.

5. Add 1ul of your second enzyme.

6. There should be a total volume of 50ul. Mix well and spin down.

7. Incubate the restriction digest at 37C for 30min, and then 80C for 20min to heat kill the enzymes. We incubate in a thermocycler with a heated lid

8. Run a portion of the digest on a gel, to check that both plasmid and part length are accurate. You may also use 2ul of the digest (20ng of DNA) for ligations.


[[BioBuilding: Synthetic Biology for Students: Lab 4]]

Lab 4: What a Colorful World


Simplifying assumptions about "the cell" are brought into question when different strains are transformed with DNA that makes them grow in colorful ways.

Acknowledgments: This lab was developed with materials from the University of Cambridge 2009 iGEM team, as well as guidance and technical insights from Drew Endy and his BIOE.44 class at Stanford University


Objectives

By the conclusion of this laboratory investigation, the student will be able to: Define and properly use synthetic biology terms: chassis, system, device, minimal cell, sensor, color generator. Define and properly use molecular genetics terms: operon, gene expression, bacterial transformation. Explain the role of chassis in synthetic biology and engineering. Conduct and interpret the results of a bacterial transformation.


Introduction


One potential use of engineered bacteria is as indicator of toxic substances. Bacterial sensing systems have been designed for arsenic and lead. Bacteria are cheap and easy to produce and store. This reduces the need for expensive and technologically complex chemical tests. The bacteria are also much more sensitive to the toxin levels. However, there is one potential drawback. The bacteria respond to the toxin metabolically. This means we may be able to detect a change in pH or other indicator of metabolism. This requires further equipment such a pH indicator. Sensors have been linked by synthetic biologists to other forms of output such as the green fluorescent protein. However, this also requires further equipment such as a fluorescent light. This reduces the practicality in impoverished areas of the world, the very areas most at risk for arsenic or lead contamination.

The 2009 Cambridge iGEM team took up the challenge to design an indicator that could be used without additional technology. They designed color generator devices that could be linked to sensors. E. coli are naturally colorless, but other bacteria make pigments and so do appear colored. The iGEM team designed “e chromi,” engineered E. coli capable of producing colors through the synthesis of pigments. One pigment they used is Violacein, a pigment produced by a handful of genes originally found in Chromobacterium violacein. These genes were re-engineered and combined to produce purple and green in E. coli. The violacein operon consists of five genes which metabolize L-tyrosine. Expression of all five genes will produce a purple pigment. However, removal of the third gene in the sequence will cause the cell to metabolize the L-tyrosine into a green pigment. These pigments are easily visible to the naked eye. This device could be linked to a biosensor for a toxin and the bacteria will turn color in response to the toxin concentration.

It's reasonable to wonder: Why didn't the team just use the Chromobacterium? Synthetic biologists like to use E. coli because it is well understood and easy and safe (if proper strains are used) to work with. But it's important to realize that this was a choice! Synthetic biologists refer to the host cell as the chassis, and just as you'd carefully design a genetic program to encode, you'd also need to carefully choose the chassis that will run it. For an engineered genetic system to function in a chassis, the chassis must supply the cell with energy, materials for protein synthesis and materials those proteins will use when they function. The chassis will take care of all the material needs to meet the engineer’s specifications. The better the chassis is understood, and the better it can provide materials for the engineered system, the better the results. By primarily using one chassis, synthetic biologists are managing complexity. A standard chassis allows engineers from many labs across the world to compare results.

Note how we also manage complexity in our everyday life. When we buy bananas or bell peppers, we simply call them bananas or bell peppers. In actuality, many varieties get mixed together in the store. But is it really important that we are aware of this when we shop? As long as the taste is similar, does it matter what variety of peppers you use? Cars, however, are a different story. A car is a highly engineered system of interconnected parts. While many of these parts are similar, they must be tailored to the size and function of the car. So, while the chassis of a truck, a GTO muscle car and a Toyota hybrid are different, so are many of the internal parts that make up the engine and the drive train. We might be able to move a radio from a truck chassis to a sports car chassis, but not much else. The car manufacturers are comfortable with this complexity and it has little effect on the user of the car. What about your computer? You can think of your computer and its operating system as a chassis, making Macs and PCs different chassis (though in computer lingo they are known as platforms). There was a time in the past when word processing files written on one platform could not be viewed or edited on the other. But interoperability was clearly needed and so the computer companies have agreed on certain standards. Through re-engineering of the programs and the chassis/platforms, users no longer get lost in the complexity. Thankfully, files written on one platform can be viewed and edited on the other.

Synthetic biologist George Church is working to further remove the complexity from engineered systems by creating what are known as minimal cells. The idea is to design a cell that contains just the minimum genome to maintain its existence. These cells will only be able to survive on special media and all of their metabolic functions will be well characterized. Another example of research into this idea was published by Craig Venter in May of 2010. His lab replaced the genome of a bacterial cell with a fully synthesized genome and were able to produce bacteria that expressed the synthetic genome. As appealling as these chassis are for synthetic biology, the work has a way to go before they can be in general use.

So, until minimal cells or synthetic cells are a viable option, researchers continue to use E coli and other domesticated cells as chassis for experiments. Mostly, the strains of E. coli that are used in research labs are one of two kinds. One strain is known as K-12 and the other B. Both strains are known to be safe and have been effectively used for genetic experiments for almost 100 years. The differences between these strains seem to be minor. Most are related to metabolism and none would seem likely to affect the color generator system. You can read about the interesting history of these strains here.

So now imagine that a group of engineers is manufacturing an arsenic sensor in E. coli. This group would like the intensity of purple color to vary as a function of arsenic level. Now imagine that a second group of engineers are also doing this but they use a different strain of E. coli. How sure can we be that the pigment will be expressed the same in a different chassis? Thinking back to our analogy with car chassis: would an engineer put a V-8 engine from a Lexus into a Mercedes chassis? Would the engine behave the same? Would the car? In this lab you will transform bacteria from two different strains of E. coli, in other words, two different chassis. Strain 4-1 is a K-12 strain, while strain 4-2 is a B-type strain. Into each strain you will insert plasmids containing violacein-pigment devices. One plasmid, pPRL, has the purple version of this device while the other plasmid, pGRN, has the dark green version. Otherwise, the plasmids are the same. Can we expect the devices to behave the same in each strain or will the chassis have an effect on the intensity of color produced? Procedure


Part 1: Preparing Strain 4-1 and 4-2 for transformation


Neither of these E. coli strains will take up DNA from the environment until they are treated with a salt solution that makes their outer membrane slightly porous. The cells will become "competent" for transformation (i.e. ready to bring DNA that's external to the cell into the cytoplasm where the DNA code can be expressed). The cells will also become fragile. Keep the cells cold and don't pipet them roughly once you have swirled them into the CaCl2 salt solution.


1. In advance of lab today, a small patch of each strain was grown for you on an LB agar petri dish. A video of this procedure is here. Strain 4-1 is a K-12 type of E. coli. Strain 4-2 is a B-type strain.

2. Label 2 small eppendorf tubes either "4-1" or "4-2".

3. Pipet 200 ul of CaCl2 solution into each eppendorf and then place the tubes on ice.

4. Use a sterile wooden dowel to scrape up one entire patch of cells (NOT including the agar that they're growing on!) labeled "4-1," and then swirl the cells into its tube of cold CaCl2. A small bit of agar can get transferred without consequence to your experiment, but remember you're trying to move the cells to the CaCl2, not the media they're growing on. If you have a vortex, you can resuspend the cells by vortexing very briefly. If no vortex is available, gently flick and invert the eppendorf tube, then return it to your icebucket.

5. Repeat, using a different sterile wooden dowel to scrape up the patch of cells labeled "4-2." Vortex briefly if possible. It's OK for some clumps of cells to remain in this solution.

6. Keep these competent cells on ice while you prepare the DNA for transformation.


Part 2: Transforming Strains 4-1 and 4-2 with pPRL and pGRN


The cells you've prepared will be enough to complete a total of 6 transformations. You will transform the purple-color generator into each strain, and also the green-color generator into each strain. You will also use the last bit of competent cells as negative controls for the transformation.


1. Retrieve 2 aliquots of each plasmid for a total of 4 samples (2x pPRL, 2x pGRN). Each aliquot has 5 ul of DNA in it. The DNA is at a concentration of 0.04 ug/ul. You will need these values when you calculate the transformation efficiency at the end of this experiment.

2. Label one of the pPRL tubes "4-1." Label the other pPRL tube "4-2." Be sure that the labels are readable. Place the tubes in the ice bucket.

3. Label one of the pGRN tubes "4-1." Label the other pGRN tube "4-2." Be sure that the labels are readable. Place the tubes in the ice bucket.

4. Flick the tube with the competent 4-1 strain and then pipet 75 ul of the bacteria into the tube labeled "pPRL, 4-1" and an additional 75 ul into the tube labeled "pGRN, 4-1." Flick to mix the tubes and return them to the ice. Save the remaining small volume of the 4-1 strain on ice.

5. Flick the tube with the competent 4-2 strain and then pipet 75 ul into the tube labeled "pPRL, 4-2" and an additional 75 ul into the tube labeled "pGRN, 4-2." Flick to mix and store them, as well as the remaining volume of competent cells, on ice.

6. Let the DNA and the cells sit on ice for 5 minutes. Use a timer to count down the time.

7. While your DNA and cells are incubating, you can label the bottoms (not the tops) of the 6 petri dishes you'll need. The label should indicate the strain you've used ("4-1" or "4-2") and the DNA you've transformed them with ("pPRL," "pGRN," or "no DNA control")

8. Heat shock all of your DNA/cell samples by placing the tubes at 42° for 90 seconds exactly (use a timer). This step helps drive the DNA into the cells and closes the porous bacterial membranes of the bacteria.

9. At the end of the 90 seconds, move the tubes to a rack at room temperature.

10. Add 0.5 ml of room temperature LB to the tubes. Close the caps, and invert the tubes to mix the contents.

11. Using a sterilized spreader or sterile beads, spread 250 ul of the transformation mixes onto the surface of LB+ampicillin agar petri dishes. A video of the procedure is here.

12. If desired the remaining volumes of transformation mixes can be plated on LB plates to show the effect of antibiotic selection on the outcome.

13. Incubate the petri dishes with the agar side up at 37° overnight, not more than 24 hours.


Next day


In your lab notebook, you will need to construct a data table as shown below. These may be provided. Also be sure to share your data with the BioBuilder community here.


1. Count the number of colonies growing on each petri dish.

 Small white colonies that are growing around the perimeter of larger colored colonies are called "satellites." They should not be counted. They grow near the central colony only after the cells there have inactivated the ampicillin that's in the petri dish agar.

 You can feel most confident in your results if there are between 20 and 200 colonies on the petri dish. Fewer than 20 and your value is affected by errors in pipeting that make large percentage differences in the outcome. Greater than 200 colonies and they become hard to count reliably. If the petri dish has many colonies growing on it, try to divide the dish into pie sections (1/4th or 1/8ths or even 1/16ths of the area), and then count a representative area. Finally, multiply the number you get for the section to get your total number of colonies. You'll still have some counting error, but perhaps less.

 Based on the number of colonies you find on each petri dish, calculate the transformation efficiency for each. Transformation efficiency is a measure for how well the cells incorporated the DNA. The units for transformation efficiency are "colonies per microgram of DNA." Each transformation used 200 nanograms (=0.2 micrograms) of DNA and you plated only 1/2 the transformation mixes on the petri dishes.


2. Record the color of the colonies you see.


 Based on these observations, do the DNA programs seems to be behaving identically in both strains for E. coli? For example, does the pPRL plasmid give the same number of transformants and the same color in both strains? What about the pGRN plasmid? If you see differences, how can you explain them? How could you test your explanations? Calculations

Here is a sample calculation for transformation efficiency Data:

100 colonies on a petri dish

0.2 micrograms of DNA used

1/2 of the transformation mix plated

Calculation:

100 x 2 = 200 colonies if all were plated

200 colonies/0.2 micrograms of DNA = 1*10^3 colonies/microgram of DNA = transformation efficiency


[[Lab Report]]



I. Introduction

Provide a brief introduction describing the field of synthetic biology. What is a color generator? How does this color generator work? How might a color generator be useful?

Briefly describe the purpose of the lab. What are we trying to do here? Presume that a reader of your lab report has not read the assignment. What is the role of the chassis? How does chassis effect the expression of a genetic system?How might synthetic engineers modify the relation between a chassis and an engineered genetic system to reduce the chassis effect on the system? Why is it important to engineer a minimal or synthetic cell? What are the advantages/concerns of engineering a minimal cell? How might we test for the differences in the chassis that may be affecting a genetic system? You may find helpful information here and here.


II. Methods


You do not have to rewrite the procedure. Explain why you did each step of the protocol.


III. Results


Present the data tables in clear format. Present drawings of each slide. Describe the results: Describe the appearance of each plate. Are the colors different? Are the colonies different in number, size and/or shape? What was the transformation efficiency for each plate? Does it differ between the strains?


IV. Discussion


Draw a conclusion: Do the color generators produce the same results in different chassis? Justify your answer. Analyze the data: Be sure to discuss how each part of the experiment and results adds to your conclusion. Are we sure that the transformation worked? What do the controls that lacked plasmid tell us? Discuss errors and other reasons for data variability. Use your results to explain why it is important for synthetic biologists to fully characterize the chassis used in an engineered system.


References


1. Hanahan D, Jessee J, and Bloom FR. Plasmid transformation of Escherichia coli and other bacteria. Methods Enzymol 1991; 204 63-113. pmid:1943786. PubMed HubMed [Hanahan91]

2. Reusch RN, Hiske TW, and Sadoff HL. Poly-beta-hydroxybutyrate membrane structure and its relationship to genetic transformability in Escherichia coli. J Bacteriol 1986 Nov; 168(2) 553-62. pmid:3536850. PubMed HubMed [Reusch86]

3. Addison CJ, Chu SH, and Reusch RN. Polyhydroxybutyrate-enhanced transformation of log-phase Escherichia coli. Biotechniques 2004 Sep; 37(3) 376-8, 380, 382. pmid:15470891. PubMed HubMed [Addison04]

4. US Patent 6,709,852 pat6709852.pdf [Bloom04]

5. US Patent 6,855,494 pat6855494.pdf [Bloom05]

6. US Patent 6,960,464 pat6960464.pdf [Jesse05] All Medline abstracts: PubMed HubMed Retrieved from "http://partsregistry.org/Help:Protocols/Competent_Cells"

Notebook



1. Week 1 (02/16-02/20)

- Monday

 Find background information on YAP1: YAP1 is a transcription activator involved in oxidative stress response and redox homeostasis made of 650 amino acids. YAP1 regulates the transcription of genes encoding antioxidant enzymes and components of the thiol-reducing pathways.

- Tuesday

 Continue research on YAP1 and YAP family.

 Read article on Gibson Assembly.

- Wednesday

 Listen to lecture presentation on Gibson Assembly.

- Thursday

 Continue research on YAP1 and YAP family.

- Friday

 Wrap up research on YAP1 and write summary.

 Begin to develop trial and error combinations for primer design


2. Week 2 (02/23-02/27)

- Monday

 Begin primer design using the translational unit kozak+mcherry and the terminator ADH1.

- Tuesday

 Use newly designed primer and attempt to construct it using Gibthon.

- Wednesday

 Look into redox-center and interaction with disulfide bonds and cysteine residues.

 Research on how mercury can affect individuals in the environment.

- Thursday

 Research GSH1 and its enzyme gamma-glutamylcycteine synthetase: GSH1 (glutathione):GHS1 is an antioxidant that plays a role in the detoxification of oxidants and toxins from a cell.

 Look into how YAP1 affects antioxidant enzymes such as gamma-glutamylcyctine synthetase.

- Friday

 Continue Research on GSH1 and gamma-glutamylcyctein syntase.


3. Week 3 (02/30-03/03)

- Monday

 Read article : The Role of Cysteine Residues as Redox-Sensitive Regulatory Switches by David Barford

- Tuesday

 Continue reading article from Monday.

- Wednesday

 Class discussion on IGEM project and progress in research.

- Thursday

 Split into groups and begin working on the team wiki.

- Friday

 Continue working on wiki notes and project overview.


4. Week 4 (03/05-03/09)

- Monday

 Continue work on project overview.

- Tuesday

 Presentation given by Dr. Goebal from IUPUI.

 Take notes and ask questions

- Wednesday

 Oxidation and ROS presentation by Forest.

- Thursday

 Split into groups and continue writing for the wiki page.

 Write about the YAP family, YAP1, GSH, and other components of the project.

- Friday

 Type notes from notebooks into word document

 Create Primer using Kozac+mCherry and ADH1; cut with ECOR1 and SPEC1.


5. Week 5 (03/12-03/16)

- Monday

 Look over and present primer to group members.

- Tuesday

 Discuss primer with class.

- Wednesday

 Split into groups and create power points on specific topics i.e. YAP1

- Thursday

 Continue working on power point.

- Friday

 Practice power points and prepare for presentation to class.


6. Week 6 (03/19-03/23)

- Monday

 Listen and take notes over YAP1 powerpoint.

- Tuesday

 Listen and take notes over Glutathione presentation.

- Wednesday

 Listen and take notes over Safety and Procedures presentation.

- Thursday

 Add information from all presentations, including human impact, to team wiki page.

- Friday

 Work on notebook.


7. Week 7 (03/26-03/30)

- Spring Break


8. Week 8 (04/02-04/06)

- Spring Break


9. Week 9 (04/09-04/13)

- Monday

 Split into groups and research on the terminator and promoter while working on the Prezi and poster board.

- Tuesday

 Continue working on Prezi and poster board.

- Wednesday

 Welcome guest speaker from Community Hospital to discuss internships.

- Thursday

 Pour plates and use serial cloner to help figure out which restriction enzymes to use.

- Friday

 Continue using serial cloner and grow bacteria on plates.


10. Week 10 (04/16-04/20)

- Monday

 Use Serial Cloner and continue to brainstorm ideas on cut sites.

- Tuesday

 Learn to ligate and cut on serial cloner.

- Wednesday

 Continue to explore ligation.

- Thursday

 Draw out sequences produced on serial cloner.

- Friday

 Draw out sequences and analyze sequences.


11. Week 11(04/23-04/27)

- Monday

 Add restriction enzymes to drawn sequence from the week prior.

- Tuesday

 Discuss and explain which enzymes are best to use and why.

- Wednesday

 Class discussion on the sequences.

- Thursday

 Guest s from community hospital network

- Friday

 Feildtrip to South Community Hospital to use DaVinci Simulator.


12. Week 12 (04/30-05/04)

- Monday

 Work on poster board,wiki page, and prezi organization.

- Tuesday

 Continue working on prezi, poster board and wikipage.

- Wednesday

 Guest Speaker: President of Community Hospital Network

- Thursday

 Dicuss internships and tomorrow's fieldtrip.

- Friday

 Class field trip to Community South simulator.


13. Week 13 (05/07-05/11)

- Monday

 Guest speaker on research careers.

- Tuesday

 Make liquid cultures of the colonies on from the streaked plates.

- Wednesday

 Guest Speaker: RN from Community Network.

- Thursday

 Throw goodbye party for Sam.

- Friday

 Work independently on research,wikipage,lab,poster and prezi.


14. Week 14 (05/14-05/18)

- Monday

 Discuss next steps in progress.

- Tuesday

 Make plates and independent study.

- Wednesday

 Grow bacteria and store plates.

- Thursday

 Continue indepenent study and prezi while bacteria grows.

- Friday

 Work on Prezi and poster board.


15. Week 15 (05/21-05/25)

- Monday

 Discuss and prepare plasmids for ligation and bacterial transformation.

- Tuesday

 Begin ligation and incubate.

- Wednesday

 Transform bacteria onto plates and incubate.

- Thursday

 Work on poster board and Prezi.

- Friday

 Analyze groth of bacteria on plates.

Results/Conclusions

Our results are inconclusive. We will provide more detail during the presentation at the Jamboree.

Safety

Hazards:

Possible Dangers to the Enviroment

Mercury is a metal that occurs naturally at low levels in rock, soil and water. Mercury is also released into the air, water and land .Most mercury pollution is released into the air and then falls directly into water bodies or onto land, where it can be washed into waterways. When mercury gets into water, bacteria can change it into a form called methylmercury, which is absorbed by tiny aquatic organisms. When fresh water and ocean fish eat those organisms, the mercury begins to build up in their bodies. When larger fish eat smaller fish, mercury can build up to high levels in the tissues of the big fish. Because it binds to the protein in fish muscles - the 'meat' of the fish - mercury cannot be removed by cooking or cleaning the fish.


Personal Injury Dangers

Although our parts have a very little chance of harming our team members, there is still that very slight chance that a problem may arise. One of the parts that we work with is mercury, which, if come in contact with the human skin, can cause great harm and in the worst case scenario, death. Therefore, we will use the proper laboratory technique to avoid any and all consequences.


Aseptic Technique

Proper aseptic technique is essential to any lab experiment. It serves to create a barrier between microorganisms in the environment and the cell cultures that one is working with. Aseptic technique is comprised of four main components: good personal hygiene, a sterile work area, sterile reagents/media, and proper disposal/ clean up.


Personal Hygiene

• Be sure to wash hands your hands properly.

• Wear closed-toe shoes.

• Wear long pants that will cover your skin.

• If you have long hair, wear it up in a ponytail.

• Wear an apron/lab coat.


Sterile Work Area

• Choose an area to work in that is free from drafts and through traffic.

• Be sure the work area is uncluttered!

• Spread materials apart to keep from bumping things and to allow for proper air flow; keep things like pipets on the right/left hand side depending on which hand you use to hold it (If you’re right-handed, keep it on the right side).

• Clean lab table before the experiment!

• Use a 70% ethanol wash.

• CAUTION: Do not use ethanol in any area if a flame will be present, since it is highly flammable.


Sterile Reagents/Media

• Wipe the outside of bottles, flasks, etc. with ethanol wash before placing them in the work environment.

• When opening a pipet, be sure not to let the tip touch anything.

• When opening bottles, don’t touch the inside of the cap; close back quickly!!

• Only use each pipet once!


Proper Disposal/Clean Up

• Wipe down all bottles/flasks again with ethanol once finished.

• Spray table with ethanol and wipe down once all materials are out of the work area.

• Contaminated waste goes in an autoclave bag! NOT THE TRASH CAN.

• If anything spills, mop it up immediately and then wipe with area with ethanol.

• Properly remove gloves.

Attributions

-Charlie Dant: research/lab

-Cheyenne Harvey:research/poster

-Rebekah Hodge: research/prezi

-Sarah Huh: research/poster

-Diamond Jones: research/wiki page

-Myka Norris :research/prezi

-Sarah Perun : research/prezi

-Patrick Steuer: research/primer design

-Amanda Weber: research/poster

-Brandon Whatley:research/lab

-Lewis Woods: research/lab

Human Practices

Mercury in the Environment

Mercury can be found in three different forms – elemental mercury, inorganic mercury, and methyl mercury. Elemental mercury is released from industrial processes, agricultural processes, household, medical products, sewage discharge, and sediment. When someone inhales elemental mercury vapor, it can cause nervous system damage. Inorganic mercury is the most common form of mercury that can be found in drinking water. Although this form of mercury can cause kidney damage, if someone is exposed to high levels, it is not considered very dangerous. However, most levels found in water are not dangerous to a human being. Inorganic mercury is actually derived from the oxidation of elemental mercury and can be found in batteries. Methyl mercury is the form of mercury that is typically found in fish. This form of mercury can bioaccumulate within the tissue of fish and humans. Although this form is not commonly found in drinking water, it can contaminate fish supply which, if ingested, can cause damage to the central nervous system and teratogenesis.

Measures Taken to Stop Mercury Pollution

In 1974, the EPA passed the Safe Drinking Water Act that set guidelines on contaminants commonly found in drinking water. In 1992, however, mercury, specifically, became regulated. Both the Maximum Contaminant Level Goal and the Maximum Contaminant Level were set at 2 parts per billion (technology can allow water suppliers to detect and remove levels of mercury that low). Monitoring of mercury levels has to take place every three months; if the level is higher than the guideline, measures must be taken to reduce these levels. There are four approved methods for removing mercury from drinking water – coagulation/filtration, granular activated carbon, lime softening, and reverse osmosis. Coagulation/filtration is the process of collecting contaminants that had coagulated in water on to a filter media. The filter media will undergoe automatic backwashing as backwash water is captured in settling tanks. The precipitate in the backwash tank sits on the bottom of the tank; then it is periodically removed for disposal via tanker truck. When settling of the precipitate occurs, a majority of the backwash supernatant is recycled through the process, which maximizes the efficiency of the system.

Fun!

We are the Warren Central Honey Badgers. Why the honey badger you ask? We are small, we are fierce, we are nasty, and we dont give a poo.