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                    WETLAB <br> -Protocol-
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<a name="myAnchor" id="myAnchor"></a><br><br><br>Protocols
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We conducted our experiments by following the protocols below.  As an official procedure, lab workers should understand the lab experiment assigned to them along with safety procedures before starting lab work. The protocols are arranged according to the order of experiments we followed.
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How to handle reagents.
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<b>1.</b>
Reagents used in our project, such as restriction reagents, must be stored in low temperature.  The reagents must be stored in the freezer when they are not used, and must be put on ice when taking them out of the freezer for an experiment. <br><br>

<b>2.</b>
Reagents should be added last to the solution, because reagents are sensitive to inactivation by pH and ionic conditions that deviate from their storage and reaction buffers. After adding reagents, the mixed solution should be mixed completely.
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Protocols to store materials and maintain <Br>
usage history of each material.
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<b>1.</b>
Reporter cell, test cell and competent cell (Top 10 invitrogen) must be kept at 4°C and frequently used reagents, reagents, DNA plasmids should be kept at −20°C in the freezer. <br><br>

<b>2.</b>
We use triple distilled water (or DDH2O) to make LB broth. Triple distilled water is kept at lab temperature (around 18 °C or lower). <br><br>

<b>3.</b>
Other materials such as yeast and NaCl are stored and maintained under the responsibility of Gachon Molecular Biology Lab. <br><br>

<b>4.</b>
We have to record the history of each material, including if plasmids/reporter cell/ test cell/ AHL have been frozen and if so, when it is used. <br><br>
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LB Medium
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<b>1.</b> To prevent contamination, we only used LB medium made within three days. <br><br>
<b>2.</b> Materials: Sodium Chloride (LB Media, Sigma), Trypton(LB Media, Sigma), Yeast Extract(LB Media Sigma), ddH2O (triple distilled water) <br><br>
<b>3.</b> Equipment: autoclave, electronic scale. <br><br>
<b>4.</b> Protocol For 200mL LB bottle <br>
1) 2 g of Sodium Chloride to a final concentration of 0.17 M  <br>
2) 2g of 1%(w/v) Bacto™ tryptone <br>
3) 1g of 0.5% (w/v) yeast extract <br>
4) ddH2O to 200 mL <br>
5) Autoclave for 20 min within 2 hours <br>
6) Keep at room temperature <br>
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LB Agar Plates and Addition of Antibiotics
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<b>1.</b> We have used LB (solidified lysogeny broth), rich growth medium for E.coli, in our experiments. <br><br>
<b>2.</b> Just before pouring the solution into petri dishes, an antibiotic can be added for resistance selection. We followed the normal working concentrations such as: <br>
- chloramphenicol: 25 μg/mL (Chloramphenicol stock is dissolved in ethanol) In case of using ampicillin: 100 μg/mL <br> 
- normal stock concentrations:1000-fold   <br><br>
<b>3.</b> Material to make LB plates: Sodium Chloride (LB Media, Sigma) Bacto™ tryptone (LB Media, Sigma) yeast extract (LB Media, Sigma) Bacto™ agar (LB Media, Sigma) ddH2O (triple distilled water) 1000x chloramphenicol or ampicillin <br><br>
<b>4.</b> LB agar preparation protocol We usually make 1liter bottle for LB Agar <br>
1) 200 mL LB prepared fresh, non-autoclaved <br>
2) 3 g agar <br>
3) Shake until all solids are dissolved <br>
4) Autoclave for 20 min within 2 hr <br>
5) Keep it cool until it reaches around 40-50 °C <br>
6) Add 200 μL of 1000x chloramphenicol and gently stir it. Be careful not to shake the bottle too long/hard so that bubbles are created. <br>
7) Pour into empty petri dishes just enough to cover the surface (~20 mL per plate). In case that bubbles are in the plate, heat the plate surface carefully with a burner only until the bubbles are burst but the solution is heated. <br>
8) Leave the plates at room temperature around one hour until it is solidified. <br>
9) Solidified plates should be turned upside down for a few hours at room temperature, then stored at 4°C. <br>
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Overnight Cultures with Antibiotics
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<b>1.</b> We have conducted overnight culture for a single bacterial strain which process needs a plate or medium with single colonies and LB containing chloramphenicol. <br><br>
<b>2.</b> Material Needed chloramphenicol: 25 μg/mL Normal stock concentrations: 1000-fold higher In case of using ampicillin: 100 μg/mL <br><br>
<b>3.</b> Protocol<br>
1) Quickly burn the neck of a bottle containing LB medium before pouring it out into a tube. Even the slightest contamination of LB will be damaging. <br>
2) Add chloramphenicol or ampicillin to give the appropriate concentration<br>
3) Scoop one colony from the plate with a sterile micropipette tip<br>
4) Immediately stick the tip into the tube containing the medium and chloramphenicol or ampicillin<br>
5) Incubate at 37°C with the shaking incubator overnight.<br>
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Agarose Gel Electrophoresis
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<b>1.</b> Agarose gel electrophoresis is used for separation and analysis of larger (>100 bases in length) nucleic acids under non-denaturing conditions. <br><br>
<b>2.</b> Analysis requires that the gel contains a DNA stain visible under UV light. Since the stain interacts with nucleic acids and is therefore potentially mutagenic, always wear nitrile gloves when working with agarose gels.<br><br>
<b>3.</b> Use protective glasses when using the UV light box.<br><br>
<b>4.</b> Material Needed<br>
Agarose<br>
1x TBE<br>
Sybr®Safe<br>
Loading dye mix<br>
DNA ladder size marker<br>
DNA samples<br><br>
<b>5.</b> Protocol: <br>
1) The gel tray must be on a level surface.<br>
2) Insert the comb into the gel tray at one end ~1 cm from the edge.<br>
3) For a 1% 150 mL agarose gel, weigh 1.5 g of agarose in a 500 mL conical flask. <br>
4) Add 150 mL 1x TBE buffer. <br>
5) To dissolve the agarose in the buffer, swirl to mix and microwave for a few minutes taking care not to boil the solution out of the flask. <br>
6) Remove the flask occasionally and check whether the agarose has dissolved completely. <br>
7) Let the agarose solution cool down.<br>
8) Once the solution is touchable, add the DNA stain. <br>
9) Check the stock concentration as the working concentration for ethidium bromide is 0.5 μg/mL while for Sybr®Safe it is simply 1x.<br>
10) Pour the gel solution into the gel tray. <br>
11) Remove any air bubbles with a pipette tip. <br>
12) Put in comb.<br>
13) The gel will solidify while cooling down to room temperature, which usually takes about 30 min.<br>
14) Running the gel by the following procedure <br>
a. Release the gel tray from the tape or casting stand. <br>
b. Place the gel tray into the buffer chamber and remove the comb carefully<br>
c. Add 1x TBE buffer until the gel is completely covered.<br>
d. Take the DNA sample (~0.2 μg) and mix with loading dye. <br>
e. Load the size marker mixed in 1x loading dye (~6 μL final volume) into a middle well.<br>
f. Load the samples into the other wells while writing down which lanes have which samples.<br>
g. Put the lid onto the buffer chamber and connect it to the power supply.<br> 
h. Run the gel at 100 V for 30–60 min. Neither of the two dyes should be run off the gel. <br>
i. Stop the run and bring the gel to a UV table to visualize the gel bands. <br>
j. Take a picture of the gel.<br>
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Gel Extraction <br>
QIAquick®Gel Extraction Kit <br>
Notes before starting
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<b>1.</b> This protocol is for the purification of up to 10μg DNA (70bp to 10kb). <br><br>
<b>2.</b> The yellow color of buffer QG indicates a pH ≤ 7.5. DNA adsorption to the membrane is only efficient at pH ≤ 7.5.<br><br> 
<b>3.</b> Add ethanol (96%100%) to Buffer PE before use (see bottle label for volume). <br><br>
<b>4.</b> Isopropanol (100%) and a heating block or water bath at 50°C are required. <br><br>
<b>5.</b> All centrifugation steps are carried out at 17,900 x g (13,000 rpm) in a convetional table-top microcentrifuge. <br><br>
<b>6.</b> Symbosl: ● centrifuge processing; ▲ vacuum processing. <br>
1. Excise the DNA fragment from the agarose gel with a clean, sharp scalpel. <br>
2. Weigh the gel slice in a colorless tube. Add 3 volumes Buffer QG to 1 volume gel (100 mg gel ~ 100μl). The maximum amount of gel per spin column is 400mg. For >2% agarose gels, add 6 volumes Buffer QG. <br>
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3. Incubate at 50°C for 10 min (or until the gel slice has completely dissolved). Vortex the tube every tube every 2-3 min to help dissolve gel. After the gel slice has dissolved completely, check that the color of the mixture is yellow (similar to Buffer QG without dissolved agarose). If the color of the mixture is orange or violet, add 10μl 3 M sodium acetate, pH 5.0, and mix. The mixture turns yellow. <br>
4. Add 1 volume isopropanol to the sample and mix. <br>
5. Place a QIAquick spin column in ● a provided 2ml collection tube or into ▲ a vacuum amnifold. To bind DNA, aply the sample to the QIAquick column and ● centrifuge for 1 min or ▲ apply vaccum to the manifold untill all the 
samples QIAquick column back into the same tube. For example volumes of 0> 800μl, load and spin/apply vacuum again. <br>
6. If the DNA will subsequently be used for sequenceing, in vitro transcruption, or microinjection, add 500μl Buffer QG to the QIAquick column and ● centrifuge for 1 min or ▲ apply vaccum. ● Discard flow through and place the 
QIAquick column back into the same tube. <br><br>
 
<b>7.</b> To wash, add 750μl Bufick column and fer PE to QIAquickcolumn and ● 
centrifuge for 1 min or ▲ apply vacuum. ●Discard flow-through and place the 
QIAquick column back into the same tube. <br><br>
<b>8.</b> Place QIAquick column into a clean 1.5 ml microcentrifuge tube.
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<b>9.</b> To elute DNA, add 50μl Buffer EB (10mM Tris•Cl, pH 8.5) or water to the 
center of the QIAquick memberane and centrifugethe colum for 1 min. For 
increased DNA concentration, add 30μl Buffer EB to the center of the 
QIAquick membrance, let the column stand for 1 min, and then centrifuge for 
1 min. After the addition of Buffer EB to the QIAquick membrance, increasing 
the incubation time to up to 4 min can increase the yield of purified DNA. <br><br>
 
<b>10.</b> If the Purified DNA is to be analyzed on a gel, add 1 volume of Loading Dye 
to 5 volumes of purified DNA. Mix the solution by pipetting up and down before loading the gel.
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Transformation Procedure
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Use this procedure to transform One Shot* TOP10 chemically competent E. coli. We recommend including the pUC19 control plasmid DNA supplied with the kit (10 pg/ μl in 5mM TrisHCl, 0.5mM EDTA, pH 8) in your transformation experiment to verify the efficiency of the competent cells. Do not use these cells for electroporation.
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<b>1.</b> Thaw, on ice, one vial of One Shot® TOP 10 chemically competent cells for each transformation. <br><br>
<b>2.</b> Add 1 to 5 μl of the DNA (10pg to 100 ng) into a vial of One Shot® cells and mix gently. Do not mix by pipetting up and down. For the pUC19 control, add 10pg (1μl) of DNA into a separate vial of One Shot® cells and mix gently. <br><br>
<b>3.</b> Incubate the vial(s) on ice for 30 mins. <br><br>
<b>4.</b> Heatshock the cells for 30 secs at 42°C without <br><br>
<b>5.</b> Remove the vial(s) from the 42°C bath and place them on ice for 2 mins <br><br>
<b>6.</b> Asceptically add 250 μl of prewarmed S.O.C. Medium to each vial. <br><br>
<b>7.</b> Cap the vial(s) tightly and shake horizontally at 37°CC for 1 hour at 225 rpm 
in shaking incubator. <br><br>
<b>8.</b> Spread 20200 μl from each transformation on a prewarmed selective plate 
and incubate overnight at 37°C. We recommend that you plate two different 
volumes to ensure that at least one plate will have wellspaced colonies. For 
the pUC19 control, dilute the transformation mix 1:10 into LB Medium (e.g. 
remove 100μl of the transformation mix and add to 900μl of LB Medium) and 
plate 25-100μl. <br><br>
<b>9.</b> Store the remaining transformation mix at +4°C. Additional cells may be 
plated the next day, if desired. <br><br>
<b>10.</b> Invert the selective plate(s) and incubate at 37°C <br><br>
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Reporter Cell Assay Protocol
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<b>1.</b> Measure out 1ml of the cells into tubes. <br><br>
<b>2.</b> Thaw out AHL on ice <br><br>
<b>3.</b> Put 3ul of AHL in test cell <br><br>
<b>4.</b> Wait 30 minutes <br><br>
<b>5.</b> Add 1ml of reporter cell to the test cell <br><br>
<b>6.</b> Wait 3 hours <br><br>
<b>7.</b> Put 200ul of the mixture into a well plate <br><br>
<b>8.</b> Put the well plate in the spectrometer to observe the results. (various independent variables such as time or the amounts of the chemical were varied in our different experiments.) (It is usually a good idea to have a control group with empty LB medium  instead of the test cell running alongside the main experiment.)
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Mini and Midi preparation <br>
QIAGEN® Plasmid Mini and Midi Kits <br>
Notes before starting
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<b>1.</b> Add RNase A solution to Buffer P1, mix, and store at 2-8°C <br><br>

<b>2.</b> Optional: Add LyseBlue® reagent to Buffer P1 at a ratio of 1:1000. <br><br>
 
<b>3.</b> Prechill Buffer P3 at 4°C. Check Buffer P2 for SDS precipitation <br><br>

<b>4.</b> Isopropanol and 70% ethanol are required. <br><br>

<b>5.</b> Symbols: ● QIAGEN Plasmid Mini Kit; ■ QIAGEN Plasmid Midi Kit <br><br><br><br>



<b>1.</b> Harvest overnight bacterial culture by centrifuging at 6000 x g for 15 mins at 
4°C. <br><br>

<b>2.</b> Resuspend the bacterial pellet in ● 0.3ml or ■ 4ml Buffer P1. <br><br>

<b>3.</b> Add ● 0.3ml or ■ 4ml Buffer P2, mix thoroughly by vigorously inverting 4-6 
times, and incubate at room temperature (1525°C) for 5 mins. If using 
LyseBlue reagent, the solution will turn blue. <br><br>

<b>4.</b> Add ● 0.3ml or ■ 4 ml prechilled Buffer P3, mix thoroughly by vigorously 
inverting 4-6times. Incubate on ice for ● 5 mins or ■ 15 mins. If using 
LyseBlue reagent, mix the solution until it is colorless. <br><br>

<b>5.</b> ●: Centrifuge at 14,000-18,000 x g for 10 mins at 4°C. Re-centrifuge if 
supernatant is not clear 
■: Centrifuge at 20,000 x g for 30 mins at 4°C. Re-centrifuge the super 
natant at 20,000 x g for 15 mins at 4°C  <br><br>

<b>6.</b> Equlibriate a QIAGEN tip ● 20 or ■ 100 by applying ● 1ml or ■ 4ml Buffer QBT, 
and allow column to empty by gravity flow. <br><br>

<b>7.</b> Apply these supernatant from step 5 to the QIAGEN tip and allow it to enter 
the resin by gravity flow. <br><br>

<b>8.</b> Was the QIAGEN top with ● 2 x 2 ml or ■ 2 x10 ml Buffer QC. Allow Buffer QC 
to move through the QIAGEN top by gravity flow. <br><br>

<b>9.</b> Elute DNA with ● 0.8 ml or ■ 5 ml Buffer QF into a clean ● 2 ml or ■ 15 ml 
vessel. For constructs larger than 45 kb, prewarming the elution buffer to 
65°C may help to increase the yield. <br><br>

<b>10.</b> Precipitate DNA by adding ● 0.56 ml or ■ 3.5 ml room temperature 
isopropanol to the eluted DNA and mix. Centrifuge at 15,000 x g for 30 mins 
at 4°C. Carefully decant the supernatant. <br><br>

<b>11.</b> Wash the DNA pellet with ● 1 ml or ■ 2ml room temperature 70% ethanol 
and centrifuge at 15,000 x g for 10 mins. Carefully decant the supernatant. <br><br>

<b>12.</b> Air dry pellet for 5-10 mins and redissolve DNA in a suitable volume of  appropriate buffer (e.g., TE buffer, pH 8.0, or 10 mM TrisCl, pH 8.5). <br><br>
</font>
</p>
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       </section>







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 <h5 style="text-align:center;">
 <font color="black">
Ligation
</font>
</h5>
<br>

<p style="text-align:center">
Quick Ligation Protocol
</p>


<div class="inner1">
<P style="text-align:left;">
 <font color="black">
<b>1.</b> Combine 50 ng of vector with a 3-fold molar excess of insert. Adjust volume to 10μl with dH2O. <br>
<b>2.</b> Add 10μl of 2X Quick Ligation Reaction Bugger and mix. <br>

<b>3.</b> Add 1μl of Quick T4 DNA Ligase and mix thoroughly. <br>

<b>4.</b> Centrifuge briefly and incubate at room temperature (25°C) for 5mins. <br>

<b>5.</b> Chill on ice, then transform or store at -20°C <br>

<b>6.</b> Do not heat inactivate. Heat activation dramatically reduces transformation efficiency. <br>
</p>
</div>

 <h5 style="text-align:center;">
 <font color="black">
Enzyme Digestion
</font>
</h5>
<br>

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 <h5 style="text-align:center;">
 <font color="black">
  Plasmid Construction Protocol
</font>
</h5>
<br>


<div class="inner0">
<P style="text-align:left;">
 <font color="black">
Last year, we isolated antibiotic resistance genes from MRSA strains from patients. First, tested the MRSA strains for antibiotic resistance, and we extracted the plasmid from those that showed antibiotic resistance, and transformed nonresistant E. coli with it. Next, we tested the transformed E. coli for antibiotic resistance. Finally, we went through PCR with the MRSA and E. coli that showed antibiotic resistance. <br><br>

For this year’s project, we used the plasmids and cells we engineered. For the main part of the experimentation, we tested the cells we engineered to make sure they worked as we made them to, and measured the various variables associated with our method. To do this, we first treated the test cell and control group with AHL, then added reporter cells to the mix. The theory was that the control group would show fluorescence, while the teste cell group would not, and the results agreed with our expectations. After that, we varied the variables to try to find significant results. <br><br>
</font>
</p>
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 <h5 style="text-align:center;">
 <font color="black">
Preliminary Work
</font>
</h5>
<br>

<div class="inner0">
<P style="text-align:left;">
 <font color="black">
We needed to increase amount of DNA vectors that were delivered from iGEM HQ. We identified what we wanted from the list of BioBrick Directory and confirmed by experiments and sequencing the lab data:
</font>
</p>
</div>
<img class="displayed" src="https://static.igem.org/mediawiki/2015/5/59/Graphics2-25.png" width="1026px">


<div class="inner0">
<P style="text-align:left;">
 <font color="black">
In the process, we found out J61100 has the characteristics of constitutive promoter and RBS. Thus we have replaced J23100 and B0030 into J61100. Also, we used K823017 instead of B0024 because it did not work as it is supposed to be and after checking with sequencing, we found that B0024 cannot be used as terminator and we instead used K823017.
</font>
</p>
</div>

 <h5 style="text-align:center;">
 <font color="black">
Material
</font>
</h5>
<br>

<div class="inner0">
<P style="text-align:center;">
 <font color="black">
The most critical material in our experiments are DNA sample received from iGEM.
</font>
</p>
</div>
</section>


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<div class="inner0">
<P style="text-align:left;">
 <font color="black">
We located target DNA from the DNA plates by looking up at the BioBrick Directory. We tried to used chloramphenicol for the most part as our antibiotic as suggested iGEM. We also added cooled mediums to ensure the antibiotic structure would not get deformed from hot temperatures when we added antibiotic. We used AHL to activate quorum sensing signal molecule.
</font>
</p>
</div>

 <h5 style="text-align:center;">
 <font color="black">
Lab Work for Plasmid Construction
</font>
</h5>
<br>


<div class="inner0">
<P style="text-align:left;">
 <font color="black">
For the project, we conducted some experiments to create the needed plasmids. To do so, we cut the necessary parts from the vectors using restriction enzymes, which takes about several hours, then ligated the target parts, transformed into a cell, and grew the bacteria in a solid LB plate for a day. After that, we conducted overnight cultures to generate larger amounts of DNA so that they can be tested by using restriction digestion techniques. Through the expansion, we could confirm that we have constructed desired DNA. After repeating the process a few times, we could create the reporter and test plasmids. <br><br>

-In order to create the plasmids, we have conducted the following methods. <br><br>
</font>
</p>
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 <h5 style="text-align:center;">
 <font color="black">
Control Experiment
</font>
</h5>
<br>

<div class="inner1">
<P style="text-align:center;">First, let's see a video showing our control experiment work:</p><br /><br /></div>

<div align="center">
<video controls>
<source src="https://static.igem.org/mediawiki/2015/0/05/Experiment_full_version.mp4" type="video/mp4">
</video>
</div>
<br /><br />

<div class="inner1">
<P style="text-align:left;">
 <font color="black">
Purpose: investigate the concentration of AHL <br>

Protocol:<br>

<b>1.</b> Measure out 11ml of LB broth into the two tubes <br>

<b>2.</b> 1ml of reporter in one of the tubes and 1ml of test cell in the other <br>

<b>3.</b> Incubate both for 3 hours <br>

<b>4.</b> Separate the 12ml of test cell into 3 4ml measures <br>

<b>5.</b> Do the same for reporter cell <br>

<b>6.</b> Add 3ul of AHL on the first tube, 4ul on the second, and 5ul on the third <br>

<b>7.</b> Separate each of the 4ml test cells into 2 2ml measures  <br>

<b>8.</b> Measure out 3 2ml of LB broth <br>

<b>9.</b> Add 3ul of AHL in one of the LB, 4ul in another, and 5ul in the last one <br>

<b>10.</b> Incubate everything for another 1 hour <br>

<b>11.</b> Add 2ml of reporter to all the non-control groups <br>
</font>
</p>
</div>



</section>

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<div class="inner1">
<P style="text-align:left;">
 <font color="black">
Purpose: find out how much AHL concentration is optimal and how much time it takes for the AHL to express GFP <br>

Protocol  <br>


<b>1.</b> Measure out 3 1ml measures of test cell <br>

<b>2.</b> Do the same for reporter cell <br>


<b>3.</b> Put 1ul of AHL in one test cell, 2ul in another, and 3ul in the third <br>


<b>4.</b> Incubate everything for 1 hour <br>


<b>5.</b> Put 1ml of reporter to each test cell <br>


<b>6.</b> Put 200ul of each mixture in one of the wells in a well plate <br>


<b>7.</b> View the results on the well plate through the spectrometer <br>


<b>8.</b> Incubate for 30 minutes in the incubator <br>


<b>9.</b> Repeat steps 7 and 8 until a significant result appears. <br><br><br>

Purpose: Find out how much time it takes for the test cell to break down AHL <br>

Protocol <br>


<b>1.</b> Mix 20ml of test cell with 100ul of AHL <br>


<b>2.</b> Put the mixture in the incubator <br>


<b>3.</b> Every 30 minutes, take out 1ml of the mixture, add 1ml of reporter and 

incubate it for 3 hours <br>


<b>4.</b> After the 3 hour incubation, put the mixture in a well plate and view the 

results every 30 minutes
 <br>
</font>
</p>
</div>
</section>

    <!-- Section #19 --!>
        <section id="maintext" data-speed="10" data-type="background">

<div class="inner1">
<P style="text-align:left;">
 <font color="black">
Protocol <br>


<b>1.</b> Prepare 6 1ml of test cells and 6 1ml of clean LB broth <br>


<b>2.</b> Add no AHL to the first test cell, 1ul to the second, 2ul to the third, 3ul to 

the fourth, 4ul to the fifth, and 5ul to the sixth <br>


<b>3.</b> Do the same for the clean LB <br>


<b>4.</b> Incubate for 30 minutes <br>


<b>5.</b> Put in well plate and view the results <br><br>

Conclusion:  <br>

It takes 3 hours for the AHL to express GFP under our conditions <br>

It takes 30 minutes to the test cell to break down AHL under our  

conditions 

3ul of AHL is optimal under our conditions
</font>
</p>
</div>

</section>

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