iGEM 2018 — Team Georgia_State

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# Team:Georgia State/Notebook

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## Lab Notebook

#### [May](#collapse1)

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#### [June](#collapse2)

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#### [July](#collapse3)

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#### [August](#collapse4)

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#### [September](#collapse5)

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#### [October](#collapse6)

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# Team:Georgia State/Experiments

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# Experiments

![](https://static.igem.org/mediawiki/2018/1/17/T--Georgia_State--ExperimentFlow.png)

# Protocols

### Plasmid Extraction

**QIAprep Spin Miniprep Kit**

1. Centrifuge 3 mL of bacterial overnight culture in two separate Eppendorf tubes at 8,000 rpm for 3 minutes at room temperature.
2. Discard the supernatant and resuspend pelleted bacterial cells in 250 μL Buffer P1 and transfer to one eppendorf tube.
3. Add 250 μL of Buffer P2 and invert each tube 5 times.
4. Add 350 μL of Buffer N3 and immediately mix by inverting the tubes 5 times.
5. Centrifuge all tubes for 10 minutes at 13,000 rpm.
6. Micropipette 800 μL of the clear supernatant into a spin column and centrifuge for 60 seconds and discard the excess liquid.
7. Add 500 μL of PB and centrifuge the spin columns for 60 seconds. Discard the flow through.
8. Add 750 μL of PE to the spin columns, centrifuge for 60 seconds, and discard the flow through.
9. Centrifuge the spin columns again for 60 seconds to remove residual wash buffer and discard the flow through.
10. Transfer the spin columns to a clean eppendorf tube and add 50 μL of EB to the center of the spin column to elute the DNA.
11. Allow the spin column to stand for one minute and centrifuge for one minute.
12. Record the concentrations for each sample.

**QIAGEN Plasmid Midi Kit**

1. Separate 100 mL of bacterial overnight culture into 5 separate 50 mL falcon tubes and centrifuge at 6,000 rpm for 15 minutes at 4°C.
2. Decant supernatant.
3. Add 4 mL of Buffer P1 to one tube, pipet mix, and transfer to another tube. Mix and transfer contents to the next tube with pelleted cells. Repeat until all tubes are combined.
4. Add 4 mL of Buffer P2 to the tube containing 4 mL of Buffer P1 and the combined resuspended pelleted cells and vigorously invert 6 times.
5. Incubate at room temperature for 3 minutes.
6. Add 4 mL of Buffer P3 and vigorously invert 10 times.
7. Incubate on ice for 15 minutes.
8. Centrifuge at 20,000 x g at 4°C for 30 minutes.
9. Once centrifuged, transfer clear supernatant to another centrifuge tube while avoiding all of the flakes on the sides and in the solution.
10. Centrifuge the tube again at 20,000 x g at 4°C for 15 minutes.
11. While this runs the equilibrate the QIAGEN-tip by adding 4 mL of QBT to the QIAGEN-tip.
12. Add the clear solution to the QIAGEN-tip and allow it to enter the resin by gravity flow.
13. Next, add 10 mL of Buffer QC to the QIAGEN-tip and allow to gravity drip.
14. Once that passes through, add 10 mL more of Buffer QC and allow to flow through.
15. Then, add 5 mL of Buffer QF and allow to flow through.
16. Add 3.5 mL of room temperature isopropanol to elute the DNA and mix. Then centrifuge for 15,000 x g for 30 minutes at 4°C.
17. Carefully remove the supernatant making sure not to disrupt the clear pellet.
18. Add 2 mL of room-temperature 70% ethanol and centrifuge for 10 minutes at 15,000 x g at 4°C. Discard the supernatant leaving as little liquid behind as possible, careful not to disrupt the clear pellet.
19. Air-dry the pellet for 20 minutes in the vent hood and redissolve in 100 μL of Buffer EB.

### Restriction Digest

**30 μL Fast Digest Restriction Digest**

1. Prepare a Fast Digest concentration cocktail with the following proportions: 1 μL Restriction Enzyme #1, 1 μL Restriction Enzyme #2, 3 μL of 10X Fast Digest Buffer, and 15 μL of diH2O.
2. Add 20 μL of this cocktail to a clean 1.5 Eppendorf tube along with 10 μL of DNA
3. Incubate at 37° C for 30 minutes.

### PCR

**20 μL Reaction**

1. Prepare a PCR concentration cocktail with the following proportions: 7 μL of diH2O, 10 μL PCR Mastermix, 1 μL of the forward primer, and 1 μL of the reverse primer.
2. Add 19 μL of the concentration cocktail into a PCR tube along with 1 μL of the DNA.
3. Place PCR tube in the thermocycler at the following generic settings:
   * 95° C for 3:00 minutes
   * 95° C for 1:00 minute
   * 52° C for 1:00 minute
   * 72° C for 1:00 minute
   * 30X (Go to Step 2)
   * 72° C for 5:00 minutes
     Lid Temperature: 105° C

### Protein Expression/Isolation

**Initial 10 mL Overnight Culture**

1. Add 10 mL of LB and 10 μL of Amp to a 50 mL falcon tube
2. Use a p10 tip to scrape the ice off of the glycerol stock and eject the tip into the falcon tube.
3. Incubated for 18 hours at 37° and 200 rpm.

**500 mL Mass Culture**

1. Dump the grown up 10 mL overnight culture from the day before into 500 mL of LB + Amp incubate at 37°C and 150 rpm for 3-6 hours.
2. Incubation time will depend on absorbance at OD 600, check periodically while the culture incubates, needs to be between 0.6 and 1.0.
3. Once the absorbance is in that range, add 500 μL of 1M IPTG to the 500 mL culture and incubated at 30°C and 150 rpm for 18 hours.

**Pelleting Cells and Resin Binding**

1. Evenly divide 500 mL mass culture into 10 different falcon tubes and centrifuge at 5,000 rpm for 15 minutes to pellet the cells.
2. Discard the supernatant from each of the tubes and resuspend the cells in 25 mL of 1X PBS with 1 mM DTT.
3. Prepare PBS from a 10X stock solution: 5 mL of 10X PBS and 45 mL of diH2O
4. Prepare 1M DTT by dissolving 0.15424 grams of DTT in 1 mL of diH2O
5. Then combine 25 mL of 1X PBS and 25 μL of 1M DTT, mix, and use as resuspension buffer above.
6. Once all of the pelleted cells are fully dissolved in 25 mL of 1X PBS with 1 mM DTT, add a Protease Inhibitor Cocktail tablet to the solution and fully dissolve. To help with this, vortex repeatedly.
7. Once it is fully dissolved, add 2 volumes of a scoopula tip full of Sigma CellLytic Express powder to the mixture and vortex.
8. Allowed the solution to sit benchside for 1 hour.
9. Sonicate the solution with short pulses while on ice.
10. Centrifuge the lysed cell solution at 14,000 rpm for 15 minutes.
11. While that runs, centrifuge 1 mL of Pierce Gluthathione Agarose at 13,000 rpm for 1 minute and remove the ethanol.
12. Wash the resin with 1X PBS with 1 mM DTT, spin down at 13,000 rpm for 1 minute, and remove the supernatant. Repeat this step 3 times
13. Then once the 1X PBS with 1 mM DTT is removed from the final wash step, resuspend the resin in the supernatant that comes from the lysed cells centrifugation from step 9.
14. Be sure to keep some of the cell debris from step 9 to run in the SDS gel and use in the Western blot.
15. Shake at 215 at 4°C overnight.

**Protein Isolation**

1. Continuing from yesterday, prepare a column by running diH2O through it.
2. Pour the chilled solution containing the resin through the column and collect the flow through. Label as flow through.
3. Pour 3 mL 1X PBS with 1 mM DTT through the spin column as a wash step and collect the flow through. Label as Wash 1.
4. Repeat step 15 twice more for a total of 3 washes.
5. Measure and record the absorbance at 280 using a UVette for each wash step.
6. Prepare a 10 mM solution of Glutathione (reduced) by dissolving 0.0307 grams in 10 mL of 1X PBS with 1 mM DTT. This will be used for elution.
7. Pour 3 mL of the elution buffer from step 18 through the column and collect the flow through. Label as Elution 1.
8. Repeat step 19 twice more for a total of 3 elutions.
9. Measure and record the absorbance at 280 using a UVette for each elution step.

### SDS Gels

**12% Tris Glycine SDS Gel**

1. Prepare the samples by combining 48 μL of the sample from protein isolation and 12 μL of 6X protein loading dye into a clean 1.5 Eppendorf tube.
2. Boil all of the samples for 5 minutes
3. Be sure to peel away the green strip of plastic located on the bottom of the gel to ensure the samples do not run diagonally and be sure the remove the comb directly vertically.
4. Load the SDS plates with 4 μL of standards and 10 μL of the samples.
5. Run at 200 mV in the prepared 1X SDS running buffer until the ladder is separated.

### Western Blot

**Transfer from Gel to Membrane**

1. Remove the gel from the SDS plates.
2. Set up transfer: Blotting paper, nitrocellulose paper, gel, membrane, sponge pad.
3. Place in transfer chamber (red to black)
4. Add prepared 1X SDS Transfer buffer to appropriate fill line along with an ice pack to help cool the solution as the transfer runs.
5. Run for 1 hour at 200 mV until the transfer is complete.

**Antibody Blotting**

1. Wash the nitrocellulose membrane with Western Blot wash buffer
2. Soak in primary antibody for twenty minutes with shaking
3. Remove primary antibody and wash with wash buffer again
4. Soak in secondary antibody for twenty minutes with shaking
5. Remove secondary antibody and wash with wash buffer once again
6. Expose to peroxide and luminol and image.

### Ligation

1. Add 6 μL of diH2O to a clean 1.5 mL Eppendorf tube, 1μL of T4 DNA Ligase Buffer, 1μL of plasmid DNA and 1μL of targeted DNA part and mix.
2. Add 1μL of T4 DNA Ligase.
3. Pipet mix the tube and incubate at room temperature for 10 minutes.

### Transformation

**Electroporation**

1. Combine 40 μL of electrically competent DH5a cells and 1 μL of ligated DNA to an Eppendorf tube.
2. Transfer the contents of the Eppendorf tube to a cuvette and lightly tap the cuvette on the table to evenly distribute the contents and get rid of air bubbles.
3. Place the cuvette into the Bio-Rad MicroPulser and deliver the electric shock.
4. Immediately after, add 900 μL SOC medium to the cuvette and micropipette mix the solution.
5. Transfer the solution from the cuvette to a shaker tube and place in a shaker at 37°C at 200 rpm for 1 hour.
6. After shaking for 1 hour, streak 150 μL of the solution onto an agar plate with the respective antibiotics.
7. Incubate plates at 37°C for 24 hours.

**Heat Shock**

1. Thaw One Shot TOP10 chemically competent cells on ice.
2. Add 2 μL of DNA sample into competent cells.
3. Incubate the cells on ice for 35 minutes.
4. After the ice incubation, place the samples into a 42° C water bath for 30 seconds.
5. Quickly take out the samples and immediately add 250µL of SOC medium to each tube.
6. Place the samples into a 37° C shaking water incubator for 1 hour at 200 rpm. After the hour, cease the shaking and allow the sample to sit in the warer until the plates are ready.
7. After shaking for 1 hour, streak 150 μL of the solution onto an agar plate with the respective antibiotics.
8. Incubate plates at 37°C for 24 hours.

### Gel Extraction

**Qiagen QIAEX II Gel Extraction Kit**

1. Run a restriction digest on the targeted DNA part using restriction enzymes and run an agarose gel for 1 hour to isolate the targeted DNA sequence.
2. Cut the targeted DNA sequence out using a razor blade, making sure to get as much DNA out while not removing too much agarose.
3. Pre-weigh empty Eppendorf tubes before adding the gel exicisions.
4. Add the gel extracts to the Eppendorf tubes and weigh again.
5. Calculate the mass of the gel using the difference of the two measurements.
6. Multiply the mass by a factor of 3 to get the volume of Buffer QX1 needed.
7. Add the respective amounts of Buffer QX1 to each of the tubes
8. Add 30 μL of QIAEX II to the samples.
9. Incubate the tubes at 50° C for 10 minutes and centrifuge.
10. Remove the supernatant and add 500 μL of Buffer QX1 to the tubes.
11. Resuspend the pellets and centrifuge for 30 seconds. Remove supernatant.
12. Resuspend the pellet in 500 μL of Buffer PE and centrifuge for 30 seconds. Remove supernatant and repeat this step.
13. Air dry pellet for 30 minutes.
14. Elute the DNA by adding 20 μL deionized water and incubate at room temperature for 5 minutes.
15. Centrifuge for 30 seconds and pipette the supernatant into a clean tube.
16. Measure and record the concentrations.

### PCR Cleanup

**Zymo Research DNA Clean & Concentrator - 5**

1. Transfer 15 μL of PCR Product into a 1.5 Eppendorf tube and add 5 volumes of DNA Binding Buffer and vortex.
2. Transfer to a Zymo-Spin Column in a collection tube
3. Centrifuge for 30 seconds and discard the flow through
4. Add 200 μL of DNA Wash Buffer to the column and centrifuge for 30 seconds.
5. Repeat Step 4 again and discard the flow through.
6. Add 30 μL of DNA Elution Buffer and incubate at room temperature for 1 minute
7. Transfer the spin column to a 1.5 mL Eppendorf tube and was centrifuge for 30 seconds to elute the DNA.

### Lab Prep

**10X TBE**

Dissolve 108 g Tris and 55 g Boric acid in 900 mL of diH2O
Add 40 mL 0.5 M Na2EDTA (pH 8.0)
Bring final volume up to 1 L and store at room temperature.

**Gel Electrophoresis**

Dissolve 1 g agarose in 100 mL of prepared 1X TBE
Cool solution down and add GelRed Nucleic Acid Gel Stain
Poor into electrophoresis chamber and add comb.
**10X SDS Running Buffer**

Dissolve 288 g of glycine, 60.4 g of Tris base, 20 g of SDS in 1.8 L of water
Adjust final volume to 2 L

**1X SDS Transfer Buffer**
Dissolve 28.8 g glycine, 6.04 g Tris base, in 1.6 L diH2O
Add 200 mL of methanol
Adjust to 2 L total volume

**10x TBS-T**
Dissolve 80.0 g NaCl, 2.0 g of KCl, 30.0 g of Tris base and 10 mL of Tween-20 into 800 mL of water
Adjust pH to 7.4 with HCl
Bring volume up to 1 liter

**Coomassie Blue Destain**

Add 400 mL of methanol and 100 mL of Acetic Acid to water
Bring total volume up to 1 liter

**Blocking Buffer**

Dilute 10x TBS-T to 1x concentration
Add 5% nonfat dry milk

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# Team:Georgia State/Results

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# Results

1. Cloning coding sequences of the parts into expression vector

a. Into Expression Vector

i. hCG Beta Subunit coding sequence was ligated into the pGEX expression vector

ii. hCG Beta 3 Loop coding sequence was ligated into the pGEX expression vector

b. Into pSB1C3 for submission

i. hCG Beta Subunit coding sequence was ligated into the pSB1C3 vector

ii. hCG Beta 3 Loop coding sequence was ligated into the pSB1C3 vector

We sent our DNA parts: hCG Beta Subunit coding part ligation into pGEX and the hCG Beta 3 Loop coding part into pGEX to Macrogen for sequencing and aligned the sequencing data against our expected constructs in SnapGene. The alignments matched, indicating a successful cloning of our coding parts into the pGEX vector.

![](https://static.igem.org/mediawiki/2018/d/d9/T--Georgia_State--recombinant_hCG_Beta_Subunit_Sequencing.jpeg)

![](https://static.igem.org/mediawiki/2018/b/b7/T--Georgia_State--Beta_3_loop_into_pGEx_sequencing.jpeg)

![](https://static.igem.org/mediawiki/2018/c/c9/T--Georgia_State--Beta_3_Loop_in_pSB1C3_Sequencing.jpeg)

![](https://static.igem.org/mediawiki/2018/1/14/T--Georgia_State--Beta_subunit_in_pSB1C3_Sequencing.jpeg)

2. Protein Expression and Isolation; hCG Antibody Recognition

a. Able to get the hCG Beta Subunit recognized by hCG antibodies

hCG Beta Subunit

The hCG beta subunit was expressed as a fusion with GST using the pGEX expression vector. After expression, and purification via glutathione column chromatography, the sample was tested for its ability to generate a positive signal on a ClinicalGuard Pregnancy Urine Test Strip. Although our test strips responded very clearly to 1.6 iU/L pure hCG purchased from Sigma-Aldrich (C1063-1VL), our recombinant fusion protein did not elicit a positive signal. We cleaved the fusion protein with HRV protease to remove the GST tag and visualized both the fusion protein and the cleavage product via Western blot using GST and hCG antibodies.

![](https://static.igem.org/mediawiki/2018/9/9a/T--Georgia_State--western_blot_and_coomassie.jpeg)

Figure 1.5 This blot contains protein elutions of the Beta 3 Loop-GST fusion protein, hCG Beta subunit-GST fusion protein, and the cleaved product of just hCG Beta subunit. This western blot was probed with anti-hCG antibodies. Lane 13 shows the hCG protein standard. Because this standard contains fully functional commercially available hCG, the band should be larger than the other recombinant hCG proteins in our other samples. The uncleaved and cleaved elutions of hCG (lanes 8 and 9) showed hCG protein. The Beta 3 Loop elutions did not contain any hCG Protein. However, the results from our coomassie blue stain leads up to think that our lysis step was not optimal, causing a large, aggregated protein mass in the cell debris (lane 6) from the expression and isolation process. The cell debris from hCG Beta subunit isolation did not contain a similar large mass, but rather produced correctly placed bands in the elutions.

We were able to successfully express the recombinant form of the hCG Beta subunit and an smaller protein which is an integral epitope present on the hCG Beta Subunit, the Beta 3 Loop. The hCG beta subunit was detected by the hCG antibodies which are similar to the non-specific polyclonal antibodies present in the Clinical-Guard Pregnancy Test strips we have used throughout the course of our project. However we did not get a positive response on the test strips with our purified protein. This could be due to very low concentrations and a lack of purity, there are still traces of other material in the elutions which could affect the antibody binding. The Beta 3 Loop was not lysed out of the cells properly, so this is just a matter of editing our experimental procedure.

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