iGEM 2021 — Team Mines

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# Team:Mines/Notebook

# Notebook

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# Team:Mines/Engineering

# Engineering Success

We went through the engineering design cycle multiple times throughout our project. First, we started with a question: how can we use genetic engineering principles to convert A-type blood to O-type blood thereby increasing the amount of O-type blood available to patients?

Our research led us to the enzymes P0DTR4 and P0DTR5, an enzyme pair that can remove the A-antigen from blood cells. This led to a new problem: how can we express these enzymes? Through some research, we landed on a couple of different ideas including using yeast or E-coli to express the three enzymes. E-coli became our organism of choice and we ordered the parts and put together a plan to insert P0DTR4 into E-coli cells and plasmids.

We used Topo TA Cloning to do this. We plated our cells onto x-gal and ampicillin plates and both blue and white E-coli colonies were expected to be observed. However, only blue colonies showed up. This caused us to need to improve our current process. Only observing blue colonies meant we were unsure if the E-coli had actually taken up the DNA fragment. Thus, we continued to improve our design, changing the concentration of gene fragments and Taq polymerase. Finally, we got a plate of both white and blue colonies leading us on to the next part of our project.

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# Team:Mines/Experiments

# Experiments

### Miniprep Protocol

![](https://static.igem.org/mediawiki/2021/1/1b/T--Mines--Miniprep.jpeg)

### Gel Electrophoresis Protocol

##### Materials

* Casting Tray
* Well combs
* Voltage source
* Gel box
* UV light source
* Microwave
* 1X TAE buffer
* Agarose
* Ethidium bromide

##### Procedure

1. Measure 1 g of Agarose
2. Mix agarose powder with 100 mL 1x TAE in a microwavable flask
3. Microwave for 1-3 min until the Agarose is completely dissolved
:   Solution should be transparent
:   Alternatively: Heat in Erlenmeyer flask on burner until solution is transparent
4. Let agarose cool down to about 50 °C
:   ~5 minutes
5. Place agarose into the gel tray with the comb in place
6. Let gels solidify for 20-30 minutes at room temperature or cool at 4 °C for 10 minutes.

##### Loading and Running Gel

1. Once solidified, place the agarose gel into the gel box.
2. Fill gel box with 1x TAE buffer until the gel is covered and then remove comb.
3. Dot a parafilm with multiple 3 µL dots.
4. Pipet 7 µL of the molecular weight ladder onto one of the dots and mix briefly.
5. Pipet this mixture into the first well of the gel making sure to not puncture the gel.
6. Repeat steps 10 and 11 for the PCR products, running the contents of both vials prepared.
7. Close the box and run the gel at 120 volts.
8. Run until the dye line is approximately 75-80% of the way down the gel.
9. When complete, turn off the power and disconnect electrodes from the power source, and then carefully remove the gel from the box
10. Visualize the gel under UV light.

### X-Gal Plate Protocol

* Prepared Xgal + Amp Plates
* Pipetted 30 mL LB broth using vacuum pipette
* Weighed 0.45 g agar
* Put agar in LB broth and heated to boiling on hot plate with stir bar
* Cooled to ~50C
* Weighed and added 6mg Xgal
* Added 3mL to solidified ampicillin to resuspend
* Added 15 uL amp to solution
* Mixed until dissolved
* Added 3mL of solution to 8 plates using vacuum pipette
* Cooled until solidified and then taped

### HindIII Digest

![](https://static.igem.org/mediawiki/2021/e/ea/T--Mines--HindIII_Protocol.png)

Note: We used 10 uL of DNA, and we doubled the amount of HindIII (used 2 uL).

### PCR Protocol

##### Materials

* Taq 2X master mix
* template DNA
* Forward Primers
* Reverse Primers
* DI water
* Ice

##### Procedure

PCR Setup

1. Resuspend primers using set amount of DI water as directed from NEB
2. Added 4μL forward primers and 4μL reverse primers
3. Added 1μL of template DNA
4. Added 100μL Taq 2X master mix
5. Added 91μL DI water
6. Separated the 200μL into 4-50μL solutions

PCR Run

1. Run 30 cycles PCR
2. Denaturation (94°C for 30s)
3. Annealing (2 @60°C for 30s, 2@ 58°C)
4. Extension (72°C for 1min)

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# Team:Mines/Results

# Results

We were able to successfully synthesize both the P0DTR4 and tPA parts. The third part due to its large size was synthesized in two parts and is soon to arrive. Both the P0DTR4 and tPA parts were successfully integrated into a TOPOTA plasmid. We were unable to successfully clone the parts into their expression plasmids (PETM6 and P406) respectively. The primary issue seems to have been unsuccessful DNA purification after digestion. Some issues with other minipreps were also experienced but the source of these issues has been fixed after switching miniprep protocols. We hope to try Golden Gate assembly with the larger P0DTR5 part, and will continue with traditional digestion ligase techniques with the other two parts.
Once we can verify we have successfully transformed cells with expression vectors we plan on using Dynabeads for protein separation and purification. We hope to preform fibrin plate analysis on both free floating tPA enzyme and compare its activity to commercially available tPA. We also hope to design microfluidic experiments to simulate more realistically blood clot lysis.
Testing of the P0DTR4 and P0DTR5 enzymes will involve the measurement of acetate concentration as a function of time for the first enzyme, and using blood type testing to verify the effectiveness of the enzyme pair.