iGEM 2023 — Team nycempirestate

![](https://static.igem.wiki/teams/4974/wiki/e-1.png)

*X** [Home](https://2023.igem.wiki/nycempirestate/index.html)
* [Team](https://2023.igem.wiki/nycempirestate/team)
  [ ]
  Team
  + [Team](https://2023.igem.wiki/nycempirestate/team)
  + [Attributions](https://2023.igem.wiki/nycempirestate/attributions%20)
* Project
  [ ]
  Project
  + [Contribution](https://2023.igem.wiki/nycempirestate/contribution)
  + [Communications](https://2023.igem.wiki/nycempirestate/communications)
  + [Description](https://2023.igem.wiki/nycempirestate/description)
  + [Engineering](https://2023.igem.wiki/nycempirestate/engineering)
  + [Education](https://2023.igem.wiki/nycempirestate/communications)
  + [Entrepreneurship](https://2023.igem.wiki/nycempirestate/entrepreneurship)
  + [Hardware](https://2023.igem.wiki/nycempirestate/hardware)
* Lab
  [ ]
  Lab
  + [Parts](https://2023.igem.wiki/nycempirestate/parts)
  + [Experiments](https://2023.igem.wiki/nycempirestate/experiments)
  + [Notebook](https://2023.igem.wiki/nycempirestate/notebook)
  + [Results](https://2023.igem.wiki/nycempirestate/results)
  + [Safety](https://2023.igem.wiki/nycempirestate/safety)
* [Human Practices](https://2023.igem.wiki/nycempirestate/human-practices)

1. [Overview](#overview)
2. [June](#june)
   * [June 26, 2023](#626)
3. [July](#july)
   * [July 9, 2023](#709)
   * [July 10, 2023](#710)
   * [July 13, 2023](#713)
   * [July 14, 2023](#714)
   * [July 15, 2023](#715)
   * [July 20, 2023](#720)
   * [July 21, 2023](#721)
   * [July 24, 2023](#724)
   * [July 25, 2023](#725)
   * [July 26, 2023](#726)
   * [July 27, 2023](#727)
   * [July 28, 2023](#728)
4. [August](#august)
   * [August 1, 2023](#801)
   * [August 2, 2023](#802)
   * [August 3, 2023](#803)
   * [August 4, 2023](#804)
   * [August 9, 2023](#809)
   * [August 10, 2023](#810)
   * [August 11, 2023](#811)
   * [August 14, 2023](#814)
   * [August 22, 2023](#822)
   * [August 23, 2023](#823)
   * [August 29, 2023](#829)
   * [August 30, 2023](#830)
5. [September](#september)
   * [September 5, 2023](#905)
   * [September 7, 2023](#907)
   * [September 14, 2023](#914)
   * [September 16, 2023](#916)
   * [September 21, 2023](#921)
   * [September 22, 2023](#922)
   * [September 28, 2023](#928)

# Overview

EmpireSpit kept a very thorough lab notebook, as we thought it crucial to precisely document all of our progress each day in the lab. Initially, we made many small errors resulting in confusing results that would’ve had no identifiable cause if not for the crisp track record we were able to look back on. Additionally, it’s much, much easier to replicate experimental steps with complete step-by-step instruction. Altogether, to stay organized, keep our timeline firm, and save time in our future recreations of experiments, it was more efficient to keep a detailed history of everything accomplished in the lab.

# June

During June, we focused on meeting requirements for getting team members into the lab and also attended an orientation.

#### June 26, 2023 (Biology Team Orientation)

###### Attendees: Christina, Seungmo, Steven

###### Time Present: 9:30-11:30 AM, 1-4 PM

Learned lab safety procedures, took a tour of the lab

---

# July

In July, we prioritized the early stages of our project. Notably, we started developing the restriction digest for our plasmid construct.

#### July 9, 2023 (Lab Setup)

###### Attendees: Christina, Steven

###### Time Present: 10 AM-12 PM

Washed dishes, organized lab space, discussed lab operations for presentation on 7/10 meeting

---

#### July 10, 2023 (Biology Team Orientation)

###### Attendees: Daniel, Heewon, Damini, Grace, Justin, Steven

###### Time Present: 9:30-11:30 AM, 1-4 PM

Learned lab safety procedures, took a tour of the lab

---

#### July 13, 2023

###### Attendees: Daniel, Heewon, Justin

###### Time Present: 12-4 PM

###### **Notes**

+ **LB Broth**

- **Homogeneous solid powder** that allows for the growth of bacterial cells.
- Contains yeast extract, sodium chloride (NaCl = Na+ and Cl- minerals / ions), and tryptone in the right ratios for the optimal growth of the bacteria.  **Yeast extract, sodium chloride, tryptone** in 2:2:1 is the most optimal ratio for growth.

+ Dissolve LB broth powder into MQH2O [**(MilliQH2O)**](https://www.emdmillipore.com/US/en/products/water-purification/type-1/5cSb.qB.89EAAAFAJqxkiQpx%2Cnav)

- **Heterogeneous solution**with some solids in the liquid → invert / inversion, x9 → autoclave, liquid 60 cycle (to boil which further dissolves and **homogenizes solution** as well as sterilizes) forming LB broth without antibiotics.

+ **Inversion / Inverting** – to make sure none of the LB broth is stuck at the bottom or the sides by shaking the solution.

+ **Autoclave** – machine used to carry out industrial/scientific processes related to elevated temperature/pressure; solution must reach temperature to clock (starting the process) let chamber preheat: 30 min. Broth cook time: 60 minutes.

- **Two probes in autoclave.**

* First, in the back is to measures the temp of air within the chamber
* Second, in the front is a silver elongated piece connected to the wire which is a probe to measure temp of solution

+ These probes are used to manage temp in autoclave.
+ Ratio to purified water = **25g /1 L milliq water** (purifies water via electric current)

+ **Microbiology Growth Curve in Four Steps:**

- **LAG:** microbes need to recover because they're frozen at -80C (absorbing nutrients). The microbes that survive are based on luck.
  Nutrients (N) >> microbes (m) OR m << N (nutrients far greater than microbe population)
- **LOG:** microbes recovered, actively dividing.
  N > m OR m < N (more nutrients to consume than microbes present)
- **PLATEAU:** microbes in homeostasis where nutrients consumed are equal to waste excreted AND the population is growing at a similar / same rate as the population dying.
  N = m OR m = N (rate of nutrient use = rate of waste)
- **DEATH:** microbe population in decline.
  N < m OR m > N (Less nutrients to consume than microbes present)

+ **Lab coats** - Take from inside the closet near Allison’s office.
  Return on Friday night so they can be washed on Monday morning.

+ **Scales & Weighing** - Ordered from most sensitive/most accurate to least sensitive / least accurate.

- Less accurate reading **scale** can measure more (25g vs 25mg) but much less accurately than the more accurate reading scale (can measure less but is much more accurate).
- Use a weight boat when measuring powder. Dispose of the weight boat in the large sharps bin.

+ **MQH2O** – Electric current that must run through water for proper purification: 18.2 megaOhms (hit the button to turn water flow on and off).

- Let the water run until 5 droplets symbol comes up, then watch the beaker level as the water fills.
- MQH2O has all impurities removed (ONLY H2O molecules), molecules (DNA, RNA etc) and minerals (ions) are removed.
- Difference from **distilled water** - water with only ions removed.

###### **Day 1 Experiment Summary:**

- We used a **scale** and a **weighboat** to measure 25g of LB Broth powder.
- We used the **MilliQ system** to measure one L of water in a **graduated cylinder**.
- We added 900 mL (.9L) of water to our 25g of LB powder.
- We utilized the **inversion** technique to mix our components 9 times (an arbitrary amount).
- We added an additional 100 mL (.1L) of water to raise the volume of water to 1L.
- We lightly sealed the media bottle, placed a slip of autoclave tape on the cap, placed both the solution and a liquid probe in an autoclave pan, and placed the temp **probe** into the **autoclave** for 1 hour to **cook / sterilize** (already had been heating for 30 minutes).

###### **Work Summary:**

- Reviewed one glove procedure.
- To prevent external particles from entering the erlenmeyer flasks, we placed aluminum foil over the top (they had already been cleaned). We did not put foil on the ones that were not cleaned. Need to clean glassware at the end of the week.
- We organized the flasks in size order on the shelves.
- Steven presented a summary in a meeting room.

---

![Lab 1](https://static.igem.wiki/teams/4974/wiki/lab1.png)

#### July 14, 2023

###### Attendees: Daniel Yi, Heewon Choi, Justin Hong, Damini Rosenblatt, Grace Lee

###### Student Time Present: 12P to 4PM

###### **Steps for Constructing Artifical Alpha Amylase**

###### **Molecular Cloning:**

- **PCR Amplification of Alpha Amylase Insert (Gene of Interest):**

* Use PCR to generate more copies of the alpha amylase insert (GOI).

- **Gel Extraction of Alpha Amylase Insert:**

* Extract the alpha amylase insert from the PCR product.

- **Linearization of Plasmid Backbone:**

* Utilize restriction enzymes (XbaI, XhoI) to break the circular plasmid backbone at the multiple cloning site (MCS).
* Perform gel extraction to obtain the linearized (broken) backbone.

- **Assembly of Plasmid:**

* Combine the alpha amylase gene (GOI) obtained in step 2 with the linearized plasmid backbone from step 3.
* Allow the GOI and backbone to anneal overnight.

- **Gel Extraction of Reconstructed Plasmid:**

* Extract the newly assembled circular plasmid.

###### **Transformation: Insertion of Plasmid into Chemically Competent Bacteria (DH5 alpha cells):**

- **Plasmid Introduction and Replication:**

* Introduce the plasmid into chemically competent DH5 alpha E. coli.
* The bacteria replicate the plasmid, producing more plasmid DNA.

- **Miniprep to Extract Plasmid DNA:**

* Perform a MiniPrep of DH5 alpha E. coli to extract and concentrate the plasmid DNA.
* Repeat this process as needed until approximately 100 nanograms of plasmid DNA is obtained.

###### **Bacterial Growth:**

- **Lag Phase:**

* Take bacteria from -80°C storage.
* Introduce the plasmid.
* Create a 1 mL day culture with shaking in LB media without Kanamycin resistance.

- **Log Phsae:**

* Take the day culture and add 4 mL to it.
* Allow overnight growth with shaking
* Subsequently, utilize the MiniPrep kit to extract and concentrate plasmid DNA.
* Make glycerol stocks for future use.

###### **Purpose of Bacterial Cultures**

- Made **LB Broth** → to grow bacteria which are currently frozen: stored -80 degrees celsius → “inactive”/”preserved” → fragile → engineered to be chemically competent - cells have been treated to have holes in bacterial cell wall:
- Purpose of Holes in Bacteria Cell Wall
  * The bacterial cell wall surrounds the bacteria
  * Cells have been treated to have holes in bacterial cell wall
  * We have the cells chemically competent, which make holes that allow plasmid DNA to go from **extracellular** (outside space) to >**intracellular** (inside space)

###### **Function of both strains of E. Coli**

- DH5alpha (DH5a for molecular cloning: backbone + endonuclease + insert = plasmid)→ makes more plasmid DNA (human alpha amylase as DNA)
- BL21 for transformation for plasmid, which came from DH5a mini prep → make plasmid DNA to alpha amylase PROTEIN

###### **Summary of each step in the alpha-amylase synthesis process**

- LB broth was made to grow bacteria(DH5alpha / DH5a, BL21)

* Stored at 80 C (frozen)
* Becomes inactive/preserved
* Engineered to be **chemically competent**

* Holes allow plasmid DNA from go from **extracellular**(outside) space to **intracellular**(inside) space
* If plasmid (DNA) was to go into the bacterial cell wall, it cannot happen without being chemically competent
* After it is made to be chemically competent, the plasmid can go into the intracellular space

- We use two different lines of e coli (DH5a and BL21)

* **DH5a**: to make more plasmid DNA(human alpha amylase as DNA)
* **BL21**: make plasmid DNA to alpha amylase PROTEIN

###### **Components of the artifical alpha-amylase**

- **Molecular Cloning**: backbone + endonuclease + insert = plasmid DNA

* Use **PCR** to make more aA gene insert
* **Gel Extraction** of aA gene(1/3)
* Use **restriction enzymes**(Xba1, Xho1) to break backbone at multiple cloning site(mcs): backbone is circular DNA that works in bacteria
* **Gel Extraction** of "broken" BACKBONE
* Assembly of aA gene/GOI with "broken" BACKBONE to make the plasmid
* **Gel Extration** of plasmid

- **Transformation of DH5a with plasmid:** makes more plasmid DNA

* Transformation: insertion of extra chromosomal DNA(the plasmid) into our chemically competent bacteria
* Take plasmid + kit(magic) = plasmid in E.coli(this produces more plasmid DNA within bacteria)
* Miniprep(kit) of DH5a to extract and concentrate plasmid DNA

- **Bacterial Growth Review**

* **Lag Phase:** take bacteria out of the -80 Celsius, then introduce the plasmid, then make 1mL, day culture w/shaking LBmedia w/o KanR. So in terms of previous steps, we add the plasmid to the DH5 alpha E. coli which produces more plasmid and then eventually we can use the log phase to add the plasmid to the BL21 E. coli.
* **Log Phase:** take the day culture + 4mL, overnight w/shaking. Afterward, we will kill all the E. coli using the miniprep kit.

#### July 15, 2023 (Full Team Meeting)

###### Attendees: In-Person Full Team Meeting

###### Time Present: N/A

Citations: 6 Points to Prove

* Blood draws expensive, invasive, unpopular
* Test kits, expensive, time-intensive/aptomer superiority
* Cortisol, Alpha-Amylase, and Specific mRNA are detectable in stress
* Inaccuracies with in-person diagnosis (therapist)
* Relationship between **stress, anxiety, depression**

#### July 20, 2023

###### Attendees: Christina Sher, Grace Lee

###### Student Time Present: 1130A to 4PM

###### Steps:

* Using LB Agar
* Pour 1000mL of water into 40g of LB agar
* Put it autoclave for LIQ60 cycle with liquid probe
* Liquid probe ensures media sterilizes and boils, which turns the agar powder into a solution
* Washed dishes using the Steris machine
* Take out the solution in autoclave
* Put the kanamycin (KAN) into the agar solution when cooled to ~160°C / cool enough to hold and pour.
* Pour into plates - preparing petri dishes

###### **Questions:**

* How much kanamycin did we add into how much LB agar?
* What is the ratio?
* Why do we wait to add the KAN?
* Where did the KAN come from?
* Where did we pour the plates?
* How did we pour the plates?
* Where did they stay that night?
* Where did they move to the next morning?
* What labels did we add to the dishes? To the bag?
* Where will the future bacterial work be done?

[Link to Document](https://drive.google.com/file/d/1Wj5nvs_-gA6lyl97Qr522Ri00qXX-uy6/view?usp=drive_link)

#### July 21, 2023

###### Attendees: Damini

###### Time Present: N/A

* Resuspend GOI in 20 uL H2O
* Determine the concentration using nanodrop → 1uL h2O to blank, 1uL DNAto read = 255 ng/uL = 5100 ng
* Make 10 uM primers for PCR for 100 uM stocks
  1 part primer: 9 parts H2O + make 10x dilution
  10 uL primer: 90 uL H2O + 10 uM dilution
* NEB says to template DNA < 1000, is 255 ng → going to dilute to 100 ng/uL
* NEB 25 uL reaction

#### July 24, 2023

###### Attendees: Daniel Yi, Grace Lee, Heewon Choi

###### Streaking for Single Colonies:

* Glycerol stock was at 4 degrees Celsius.
* Stabbed in the bacteria room with pipette tip.
* Streaked the bacteria onto LB (nutrients to grow bacteria) Agar (growth matrix/foundation) with Kan (kanamycin, antibiotic, selection factor; screens out any bacteria that DOES NOT contain kanR gene: a protein that will enzymatically degrade the Kan: carbohydrate antibiotic)
* Protect our bacteria of interest that contain the plasmid.
* Bacteria vs. Die because no plasmid that has the KanR gene.

###### Today:

* The glycerol stock (contains the plasmid PET-28a(+)) stored in the freezer at 4 degrees Celsius is metabolically inactive/inert (does that multiply).
* We took pipette tips and left for the freezing room.
* We went to the freezing room to get the LB Agar with Kan in the Petri dishes that were prepared last week.
* We took it to the bacteria room and stabbed the pipette tip into the glycerol stock for the single colony growth technique Streaking for single colonies.
* We streaked the glycerol stock onto the Petri dish that has LB Agar with Kanamycin. This allows the bacteria in the glycerol stock to become metabolically active/inert (allows it to multiply).

###### DNA and RNA:

* DNA makes a copy of its instructions into mRNA (leaves the nucleus in human and plant cells and goes to the endoplasmic reticulum). In bacteria, DNA is transcribed in the cytosol.
* The smallest living entity is the cell in biology, in bacterial, animal, and plant cells.
* 3 RNA nucleotides = 1 codon = 1 amino acid.
* 4 exceptions to this rule.

###### Additional Information:

* Streaking for Single Colonies:

+ Glycerol stock was at 4C.
+ Stabbed in bacteria room with pipette tip.
+ Streaked the bacteria onto LB agar with kanamycin.
+ Put it in 37C incubator (not liquid stock so not put in the shaker).

* LB: nutrients for growth.
* Agar: growth matrix/foundation.
* Kanamycin: it's an antibiotic → selection factor → screens out any bacteria that does not contain KanR gene.
* KanR is a protein that will enzymatically degrade Kan (carbohydrate) antibiotic. This is to protect our bacteria of interest that contain the plasmid.
* The point of streaking is to wake up the glycerol stock that is inert. We have to wake them up.
* We are streaking to get single colonies of bacteria.
* Overnight Culture:

+ Take out the colonies and put into LB broth to make the bacteria multiply more.
+ Use Miniprep to extract plasmid.

* PCR for gene of interest → gel extraction for gene of interest → gibson cloning → DH5a cells.
* We are engineering our own plasmid to add to DH5a cells.
* Store-bought plasmid (no gene of interest) cut it open → linearized plasmid (no gene of interest) (Gibson cloning) → homemade plasmid with gene of interest.
* Glycerol stock (E. coli #1).
* Plasmid backbone.
* DH5a (E. coli #2).
* Linearized backbone + gene of interest as DNA.
* BL21 (E. coli #3).
* Linearized backbone + gene of interest as protein.
* DH5a (DNA) → miniprep → homemade plasmid (DNA) → BL21 (DNA) → central dogma of homemade plasmid (DNA A=T G=C) → transcription to RNA (A=U, G=C) → translation to protein (amino acids) → isolate human alpha-Amylase.

---

![Lab 9](https://static.igem.wiki/teams/4974/wiki/lab9.jpeg)

![Lab 10](https://static.igem.wiki/teams/4974/wiki/lab10.jpeg)

#### July 25, 2023

###### Attendees: Damini

###### Time present: 12:30 - 3:15

###### Gel Electrophoresis:

* Create a 1x TAE dilution

+ 10% 10X TAE + 90% MQH2O
+ 100 mL 10X TAE + 900 mL water = 1000 ml (1L) 1X TAE
+ Add 100 mL of TAE and 900 mL of water to make the dilution

* Make agarose gel

+ 1% gel = 1% agarose + 100 mL fresh TAE + 5 uL SYBR dye
+ 1g agarose + 100 mL fresh TAE
+ Microwave 45s → swirl → Microwave 45s → + 5 uL SYBR dye → pour in cold room → wait until solidified

* Run gel 150 mV —> 45 min

+ 5 uL 6X loading dye + 25 uL pcr reaction → load 30 uL per well
+ Load 15 uL of ladder in the first (farthest left) well

###### Ladder-Sample-s-s-s-s

+ DNA. d w d water
+ 1 2 3 7 8

###### Gel Extraction:

* [Your gel extraction steps go here]

#### July 26, 2023

###### Attendees: Justin Hong, Heewon Choi

###### Creating an Overnight Culture (from Addgene):

* [Inoculate Bacterial Culture Protocol](https://www.addgene.org/protocols/inoculate-bacterial-culture/)

* Add KAN to LB broth (culture media) 100 μl 50 mg/mL stock to 100 mL LB broth (Steven’s email). Using these ratios, we combined 900 μl KAN:900 ml LB Broth (this is what we have). Then we inverted and relabeled LB Broth
* Moved the solution of KAN and LB Broth to the bacteria room. Took bacterial culture out of the 37° C incubator. Added 50 mL of LB KAN to each erlenmeyer flask. Gently scraped bacteria from plate using pipette tip, and added one to each erylenmeyer flask. Added a foil cap and a label to each erlenmeyer flask, and placed each of the flasks to the shaking incubator at 37°C. Sealed the plates containing the bacterial culture and placed them in 4°C.

---

![Lab 11](https://static.igem.wiki/teams/4974/wiki/lab11.jpeg)

#### July 27, 2023

###### Attendees: Grace Lee, Damini Rosenblatt

###### Steps:

* Pour 45 ml of water into one conical tube for balance.
* Pour 45 ml of bacteria in another tube and pour 50 ml of bacteria into two other tubes.
* Put into a centrifuge to separate bacteria from growth media.
* All the bacteria that was mixed with the growth media was separated to the bottom of the conical tube.
* Pour out growth media opposite of the slope of the bacteria so it doesn’t get poured out.
* Put bacteria into the freezer.
* Pour 40 ml of ethanol to 2 Buffer PE Wash buffer 10ml concentrate.
* Add 3 volumes of Buffer QG to 1 volume gel (100 mg gel ~100 pl). We have 726 mg of gel and multiply by 3 to get 2,178 volume (we used roughly 2,000 because tubes were too small).
* Put gel with buffer QG into the incubator. Incubate at 50°C for 10 min (or until the gel slice has completely dissolved). Vortex the 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 10 μl 3 M sodium acetate, pH 5.0, and mix. The mixture turns yellow.
* Add 1 gel volume isopropanol to the sample and mix.
* Place a QIAquick spin column in a provided 2 ml collection tube or into a vacuum manifold.
* To bind DNA, apply the sample to the QIAquick column and centrifuge for 1 min or apply vacuum to the manifold until all the samples have passed through the column. Remove flow-through and place the QIAquick column back into the same tube.
* Put 750 μl buffer PE into QIAquick and centrifuge for a minute or so to clean. Remove the flow through and place QiAquick back into the same tube.
* Place the QIAquick column into a clean 1.5 ml microcentrifuge tube.
* Remove the DNA by adding 50 μl water to the center of the QIAquick membrane and centrifuge the column for 1 min.

![Lab 12](https://static.igem.wiki/teams/4974/wiki/lab12.png)

![Lab 13](https://static.igem.wiki/teams/4974/wiki/lab13.png)

![Lab 14](https://static.igem.wiki/teams/4974/wiki/lab14.jpeg)

#### July 28, 2023

###### Attendees: Heewon, Daniel, Justin

###### Concentration of our plasmid from our miniprep:

* [Qiagen Miniprep Concentration Protocol](https://www.qiagen.com/us/resources/download.aspx?id=56b0162c-23b0-473c-9229-12e8b5c8d590&lang=en)

---

![Lab 15](https://static.igem.wiki/teams/4974/wiki/lab15.jpeg)

#### August 1, 2023

###### Attendees: Heewon, Grace

###### Gel Electrophoresis:

* Damini already made the TAE
* Make agarose gel (original instructions)

+ 1% gel = 1% agarose + 100 mL fresh TAE + 5 uL SYBR dye
+ 1g agarose + 100 mL fresh TAE
+ Microwave 45s → swirl → Microwave 45s → + 5 uL SYBR dye → pour in cold room → wait until solidified
+ We are going to use 75 mL fresh TAE instead

* Run gel 150 mV —> 45 min

+ 5 uL 6X loading dye + 25 uL pcr reaction → load 30uL per well
+ load 15 uL of ladder in the first (farthest left) well

###### Ladder-Sample-s-s-s-s

* DNA. d w d water
* 1 2 3 7 8

---

![Lab 16](https://static.igem.wiki/teams/4974/wiki/lab16.jpg)

![Lab 17](https://static.igem.wiki/teams/4974/wiki/lab17.jpg)

#### August 2, 2023

###### Attendees: Heewon, Justin

###### Restriction digest

* \*All bands @ 6000nt/6kb
* 6kb = plasmid
* \*\*MISSING 5.5kb PRODUCT
* 5.5kb = broken/cut/linearized plasmid
* \*\*\*MISSING .5 kb product
* .5kb = excised MCS

We know the Restriction Digest failed because in the image of the gel on 8/1/23, there are no signs of the 5.5kb product or the .5kb product meaning the restriction enzymes did not work.

###### A

* One thing we can try is to test different cut sites to make sure the manufacturer didn’t make any errors.
* We will be trying:

+ Nde I(146bp)
+ Hind III(675bp)

* We would expect a product of 529 bp and 5302 bp

###### B

* Mix + match enzymes

###### C

* Vary the amount of plasmid we’re asking to cut

+ Is there too much DNA for the enzymes to cut
+ We can try this today

![Lab 20](https://static.igem.wiki/teams/4974/wiki/lab20.png)

#### August 3, 2023

###### Attendees: Damini

###### Finish gel extraction:

* Placed gel on eyebright and take photo
* Read ladder then cut out bands containing bacteria without cloning site
* Add 3 volumes buffer to 1 volume gel

|  |  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- | --- |
| 350mg | 652 mg\* | 411mg | 340mg | 384mg | 324mg | Total volume: 1809 |
| 1050uL (1000 + 50) | 1956uL | 1233uL (1000 + 233) | 1020uL (1000 + 20) | 1152uL (1000 + 152) | 972uL (972) |
| 1400 | 2608 | 1644 | 1360 | 1536 | 1296 | 7236 9045/800=11 |

+ **\*sample too large for column, eliminating from gel extraction. Put in fridge**

* Combine into 1 large tube
* Pipette 800uLinto the spin columns
* Place in centrifuge for 1 minute at top speed
* Separate spin column from collection tube and discard flowthrough
* Place in centrifuge and spin 1 min top speed again for residual flowthrough
* 750 uL buffer PE
* Repeat steps h and I
* Separate spin column from test tube and placed in clean tube
* Pipette 30 uL of buffer TE on each spin column membrane and spun in centrifuge
* Discard spin column and combined all samples into a single sample
* Nano Dropped to find concentration: 4.9 ng/uL
* Place in yellow box in freezer

#### August 4, 2023

###### Attendees: Justin

---

![Lab 21](https://static.igem.wiki/teams/4974/wiki/lab21.jpg)

![Lab 22](https://static.igem.wiki/teams/4974/wiki/lab22.jpg)

![Lab 23](https://static.igem.wiki/teams/4974/wiki/lab23.jpg)

![Lab 24](https://static.igem.wiki/teams/4974/wiki/lab24.jpg)

#### August 9, 2023

###### Attendees: Justin

---

![Lab 25](https://static.igem.wiki/teams/4974/wiki/lab25.jpg)

![Lab 26](https://static.igem.wiki/teams/4974/wiki/lab26.jpg)

![Lab 27](https://static.igem.wiki/teams/4974/wiki/lab27.jpg)

![Lab 28](https://static.igem.wiki/teams/4974/wiki/lab28.jpg)

#### August 10, 2023

###### Attendees: Heewon, Justin

---

![Lab 29](https://static.igem.wiki/teams/4974/wiki/lab29.jpg)

![Lab 30](https://static.igem.wiki/teams/4974/wiki/lab30.jpg)

#### August 11, 2023

###### Attendees: Damini, Steven

###### Student Time Present: 1:30PM to 3PM

###### Notes

* Make a 1% gel (8-1)
* Do NEB restriction digest.

###### Using modifications follow procedure (8-1)

+ 3 rxns (1 uLDNA, 37\*C, 30 min ( between 15-60 min))

- Uncut control, 0 enzymes, same master mix
- Xho 1 only = 1 enzyme, Xho 1
- Xba 1 only = 1 enzyme, Xba 1

* 1 Rxn;

+ H2O:36.6 uL
+ Buffer:5uL
+ Enzyme 1: 1uL
+ Enzyme 2: 1uL
+ 1 ug DNA: 6.4 uL

+ 0 enzymes —> H2O: 38.6 uL (+2) E1: 0 (-1) E2: 0 (-1)
+ 1 enzyme —> H2O: 37.6 uL (+1) E1: 1 uL E2: 0 (-1)
+ 1 enzyme—> H2O: 37.6 uL (+1) E1: 0 (-1) E2: 1 uL
+ 2 enzymes —> H2O: 36.6 uL E1: 1 uL E2: 1 uL

* Protocols
  + |  |  |  |
    | --- | --- | --- |
    |  | 1 rxn | 3.5 rxn |
    | H2O | 38.6 uL | 135.1 uL |
    | Buffer | 5 uL | 17.5 uL |
    | Xho 1 | 0 | 0 |
    | Xba 1 | 0 | 0 |
    | DNA | 6.4 uL | `—` |
    | Total | 50 uL | (43.6 uL mix + 6.4 uL DNA per reaction) |
  + |  |  |  |
    | --- | --- | --- |
    |  | 1 rxn | 3.5 rxn |
    | H2O | 37.6 uL | 131.1 uL (limit by pip) |
    | Buffer | 5 uL | 17.5 uL |
    | Xho 1 | 1 uL | 1 uL |
    | Xba 1 | 0 | 0 |
    | DNA | 6.4 uL | `—` |
    | Total | 50 uL | (43.6 uL mix + 6.4 uL DNA per reaction) |
  + |  |  |  |
    | --- | --- | --- |
    |  | 1 rxn | 3.5 rxn |
    | H2O | 37.6 uL | 131.3 uL |
    | Buffer | 5 uL | 17.5 uL |
    | Xho 1 | 0 | 0 |
    | Xba 1 | 1 uL | 1 uL |
    | DNA | 6.4 uL | `—` |
    | Total | 50 uL | (43.6 uL mix + 6.4 uL DNA per reaction) |
  + |  |  |  |
    | --- | --- | --- |
    |  | 1 rxn | 3.5 rxn |
    | H2O | 36.6 uL | 128.2 uL |
    | Buffer | 5 uL | 17.5 uL |
    | Xho 1 | 1 uL | 1 uL |
    | Xba 1 | 1 uL | 1 uL |
    | DNA | 6.4 uL | `—` |
    | Total | 50 uL | (43.6 uL mix + 6.4 uL DNA per reaction) |

###### Reasoning for reaction:

* Goal: express human alpha amylase protein (HAA)

+ To do that we need to put HAA in plasmid
+ To do that we need to cut out the multiple cloning site (MCS) using xho 1 and Xba 1 to cut out MCS then insert the gene of interest (GOI) to create the new plasmid

* Our problem: the MCS won't get out of the plasmid

+ Is it the DNA?

- Manufacturer: solutions- sequencing or PCR(shorter)
- Concentration

+ Is it the enzymes?

- If enzymes worked: we get MCS (~650bp) and plasmid (~6000 LP); not currently happening
- Both enzymes

#### August 14, 2023 (Biology Team Orientation)

###### Attendees: Benjamin Li, Charlotte

###### Time Present: 9:30-11:30 AM, 1-4 PM

Learned lab safety procedures, took a tour of the lab

---

#### August 22, 2023

###### Attendees: Daniel, Steven

###### Student Time Present: 12PM to 3:45PM

* Discussed the progress of our project.
* We failed to cut our plasmid at 6650 bp (base-pairs) 3 times. The restriction enzymes are supposed to cut the plasmid, but NEB responded declaring that their enzymes were not the source of the problem. Thus, we wondered if the issue is with the plasmid.
* OUR OBJECTIVE: To determine if the issue in cutting lies with the plasmid.
* EXECUTION:

1. Contact Addgene, who designed our plasmid
2. Experiment: Sequence the plasmid

* Began to take lab notes in a notebook Steven provided the team with. Daniel took handwritten notes.
* Discussed the concept of sequencing: A technique used to determine the primary structure of an unbranched polymer.
* In our project, sequencing will determine the order of bases/nucleotides of our plasmid.
* Ordered a shipment to plasmidsaurus in order to check whether there is an issue with the plasmid.

---

![Lab 31](https://static.igem.wiki/teams/4974/wiki/lab31.jpg)

###### Daniel taking notes

![Lab 32](https://static.igem.wiki/teams/4974/wiki/lab32.jpg)

###### Daniel with the readied shipment to be made to plasmidsaurus for determining whether there is an issue with the plasmid.

![Lab 33](https://static.igem.wiki/teams/4974/wiki/lab33.jpg)

![Lab 34](https://static.igem.wiki/teams/4974/wiki/lab34.jpg)

#### August 23, 2023

###### Attendees: Hee won, Steven

###### Student Time Present: 6PM to 8PM

###### Sequencing of Plasmid

* Plasmidsaurus was unable to sequence the plasmid, suggested DnAse based degradation.
* Spoke with Ozge → plan is to make new mini prep of plasmid.
* Check if the incubator can do 2 mL overnight cultures → color rack on black rubber mat @ 37 Celsius.
* 8 x 2 mL overnight culture.

#### August 29, 2023

###### Attendees: Daniel, Steven

###### Student Time Present: 7PM to 7:30PM

###### Overnight Culture

8 x 2 mL @ 37 Celsius shaker.

#### August 30, 2023

###### Attendees: Hee won, Steven

###### Student Time Present: 7PM to 7:30PM

###### Overnight Culture

8 x 2 mL → 6X frozen pellets @ -20 Celsius.

#### September 5, 2023

###### Attendees: Justin, Rin, Steven

###### Student Time Present: 4PM to 8:30PM

###### Mini-prep

* New Qiagen kit → 24 mL 100% ELDH.
* 6X pellet → 300 uL @ 44.5 ng / uL → @ -20 Celsius.
* → kit suggested 2 ug / ~ 2mL culture, yield seems correct (44 x 50 x 6 = 13,200 ng).
* Could this have worked?
* → put two old samples to the side
* - will do digest @15 min w/ 1 ug DNA (22.5 uL plasmid)
* - do A and D from 08/11/2023 (DR)

#### September 7, 2023

###### Attendees: Hee won, Justin, Daniel, Steven

###### Student Time Present: 6PM to 8:45PM

###### Restriction Digest

Following 08-11 protocol for sections and A and D.

#### September 14, 2023

###### Attendees: Hee won, Steven

###### Student Time Present: 6PM to 8:30PM

###### Checking the Bacteria

Current bacteria grows on Kanamycin (KAN) plates BUT does NOT get cut by XbaI and XhoI.

Is this the bacteria that we really want?

Growing the bacteria (from addgene 135521) on empty plates, KAN plates (single line), AMP plates (double line).

#### September 16, 2023

###### Attendees: Daniel, Hee won, Steven

###### Student Time Present: 7PM to 8PM

###### Agar Plates

Made AMP Agar plates → two stripes.

#### September 17, 2023

###### Attendees: Daniel, Steven

###### Student Time Present: 7PM to 8PM

###### Streaking Plates

Streaking plates with NEW stab of backbone

#### September 21, 2023

###### Attendees: Justin, Hee won, Steven

###### Student Time Present: 7PM to 7:45PM

###### Overnight Cultures

Overnight cultures 6\*3mL

#### September 22, 2023

###### Attendees: Daniel, Justin, Steven

###### Student Time Present: 6PM to 8:30PM

###### Mini-prep for Plasmid Backbone

Followed protocol from 08-11. Generated 15.3 ng/uL in about 400 uL.

#### September 28, 2023

###### Attendees: Daniel, Justin, Steven

###### Student Time Present: 6PM to 8:30PM

###### Restriction Digest

Follow the protocol from 09-07. Determine 1 µg of DNA.

---

![](https://static.igem.wiki/teams/4974/wiki/icons/spotify-512.png)
![](https://static.igem.wiki/teams/4974/wiki/icons/instagram-5-xl.png)
![](https://static.igem.wiki/teams/4974/wiki/icons/youtube-4-xl.png)

© 2023 - Content on this site is licensed under a [Creative Commons Attribution 4.0 International license](https://creativecommons.org/licenses/by/4.0/).

The repository used to create this website is available at [gitlab.igem.org/2023/nycempirestate](https://gitlab.igem.org/2023/nycempirestate).

{% extends "layout.html" %}
{% block title %}Engineering{% endblock %}
{% block lead %}{% endblock %}
{% block page\_content %}

1. [Engineering Overview](#overview)
2. [Design](#design)
3. [Build](#build)
4. [Test](#test)
5. [Learn](#learn)

## Engineering Overview

This year, our team incorporated the engineering design process to identify aptamers and design/construct a plasmid to express alpha-amylase. Within our design process, we also used aptamers to identify a relative quantity of alpha-amylase protein. From this, we learned about some of the challenges of performing restriction digests, as well as what we would have done differently.

## Design

As high school students, all of us are subject to high amounts of stress dealing with the pressure of preparing for difficult exams, college preparation, and lengthy homework assignments as well as the difficulties caused by the pandemic. Methods of detecting stress exist but most are either extremely time-inefficient and/or expensive. This intersection between stress and patient care inspired us to find a brand new method of detecting stress that can be easily accessible and administered.

We proposed saliva as the biological medium to detect stress because it is non-invasive, as there is no need to extract blood or cause unnecessary harm to the body. Our next step was to choose biomarkers in the saliva that would act as the best indicator of stress. Cortisol and alpha-amylase were easy to choose because they have been widely acknowledged among the scientific community as salivary stress biomarkers, with cortisol even being coined “the gold standard marker of stress.”

In our research for selecting salivary biomarkers, we came across an article published by Dr. Conrad Wiegand, “Stress-associated changes in salivary microRNAs can be detected in response to the Trier Social Stress Test: An exploratory study” ([Wiegand 2018](https://www.nature.com/articles/s41598-018-25554-x)), which found significant associations with stress in three microRNAs: miR-20b, miR-21, and miR-26b. Our lab advisors Steven Reddy and Özge Begli suggested we use aptamers as a method for detecting cortisol and alpha-amylases, a relatively newer and less researched detection method that is designed to bind to target molecules. These aptamers emit fluorescence as an indication of the presence of those target molecules.

## Build

Our team engineered alpha-amylase plasmid DNA containing two components: the human alpha-amylase insert gene (gene of interest, GOI) and the excised pET-28a(+)-FGF2-G3 plasmid (linearized backbone).

We bought the human alpha-amylase insert gene and used PCR (polymerase chain reaction) to acquire copies of the human alpha-amylase insert gene to use for the construction of the recombinant plasmid. For the linearized backbone, we first grew an overnight bacterial culture containing colonies with the plasmid PET-28a(+) using the Addgene protocol on inoculating a liquid bacterial culture. Once we got enough bacterial colonies as a result of the overnight culture, we isolated the plasmid PET-28a(+) in the colonies using the directions from the QIAprep Spin Miniprep Kit.

Afterwards, we used the enzymes XbaI and XhoI on the plasmid PET-28a(+) that we obtained through a process known as a restriction digest, in which the two restriction enzymes excised (cut out) a piece of the plasmid backbone that we would eventually replace with the human alpha-amylase insert gene. We used gel extraction to isolate the broken fragments of the plasmid backbone. Then we put together the human alpha-amylase insert gene with the linearized backbone to form the recombinant plasmid using a Gibson assembly kit.

After cloning and isolating our recombinant plasmids made from the GOI and linearized backbone through the use of DH5a E. coli cells, we transformed them into BL21 E. coli cells. BL21 cells are a commonly used bacterial strain for the production of recombinant proteins, including alpha-amylase. After transformation, the BL21 bacterial culture secreted alpha-amylase proteins.

## Test

After creating the artificial alpha-amylase, we wanted to test the aptamer detection method using saliva models containing differing concentrations of alpha-amylase. Initially we planned on using fluorescence-based aptamers that would first bind to target proteins in an assay and emit a certain level of light intensity depending on the concentration of that target protein. However, as we continued our experiments, it became apparent to us that these types of aptamers were not suitable for detecting alpha-amylase because of the requirements of needing a fluorescent plate reader, leading us to look at aptamers that did not emit fluorescence. These aptamers could only be used to assess the concentration of the aptamer and protein complex by running the assay in a polyacrylamide gel electrophoresis, and depending on the distance the molecules travel, we could determine how much of the target protein was in the solution/sample.

## Learn

During our lab work, one of the problems that we encountered was performing the restriction digest to insert the PCR product and attempting to make our Alpha-Amylase with an unpopular plasmid (pET-28A), running into situations where it simply would not work. One of the major downsides of this was the lack of resources, such as prior research using the same plasmid, to turn to when we ran into such issues due to our plasmid’s unpopularity. One thing we could have done was to work with a more reliable and historically tested plasmid instead.

{% endblock %}

{% extends "layout.html" %}
{% block title %}Experiments{% endblock %}
{% block page\_content %}

1. [Protocol Overview](#overview)
2. [1. pET-28a(+)-FGF2-G3ﬁ with XbaI and XhoI](#1)
   * [1a. Restriction Enzyme Digestion](#1a)
   * [1b. Gel Extraction](#1b)
3. [2. Alpha Amylase](#2)
   * [2a. PCR Amplification](#2a)
   * [2b. Gel Extraction](#2b)
4. [3. Assembly](#3)
5. [4. Transform Assembly](#4)
6. [5. Growth of Clones](#5)
7. [6. Plasmid Isolation](#6)
8. [7. Verification of Cloning](#7)
9. [8. Verification of Transformation](#8)
10. [Our Findings](#result)
11. [References](#references)

## Protocol Overview

![](https://static.igem.wiki/teams/4974/wiki/experiments/workflow.jpg)

## 1. pET-28a(+)-FGF2-G3ﬁ with XbaI and XhoI

#### 1a. Restriction Enzyme Digestion of pET-28a(+)-FGF2-G3ﬁ with XbaI and XhoI

You need a linearized backbone for cloning of the insert via Gibson assembly. pET-28a(+)-FGF2-G3fi is
the name of the plasmid that you are going to use as the backbone. pET-28a(+)-FGF2-G3fi has a
multiple cloning site containing XbaI and XhoI restriction sites and in between you will clone the
codon optimized human alpha amylase sequence.

![](https://static.igem.wiki/teams/4974/wiki/experiments/picture1.png)

Figure 1: Sequence for Plasmid Backbone (pET-28a(+)-FGF2-G3ﬁ with XbaI and XhoI)

#### 1b. Gel Extraction If FGF2 transgene is there, you should see two bands following restriction enzyme digestion. You need the one with a bigger size, backbone, which is around 5 kbps. You should extract DNA from the band giving the desired signal. You should measure the concentration, A260/280 and A260/230 values and keep a record of those. ![](https://static.igem.wiki/teams/4974/wiki/experiments/picture2.png) Figure 2: Expected Results after Restriction Digest

## 2. Alpha Amylase

#### 2a. PCR Amplification of Alpha-Amylase

You will receive human alpha amylase sequence as a lyophilized gene fragment. You should dissolve
it in the proper solvent (i.e. ddH 2 O or TE) by following the instructions. To increase the efficiency and
the amount of the product, you should conduct a PCR assay using following primers:

* alphaamylase\_cloning\_forward: 5’ CTATAGGGGAATTGTGAGCGG 3’
* alphaamylase\_cloning\_reverse: 5’ TAGCAGCCGGATCTCAGTG 3’

#### 2b. Gel Extraction

The size of the PCR product should be around 1.6 kbps (left hand homology + alpha amylase + right
hand homology). Following agarose gel electrophoresis, you should measure the concentration,
A260/280 and A260/230 values and keep a record of those.

## 3. Assembly

|  | 3:1 (Insert:Vector) Ratio |
| --- | --- |
| Backbone (~5 kbp) | X ul (50 ng) |
| Insert (~1.6 kbp) | Y ul (50 ng) |
| NEBuilder HiFi DNA Assembly Master Mix | 10 ul |
| Deionized H2O | 10 - (X+Y) ul |

## 4. Transform Assembly

Transform the assembly product into DH5a by following the transformation protocol

## 5. Growth of Clones

Grow clones on selective agar plates with antibiotic and selection of some successful clones for verification

+ You can take -80 o C stocks at this stage to have backups of successful clones.

## 6. Plasmid Isolation

Following the instructions of QIAprep Spin Miniprep Kit

## 7. Verification of Cloning

You should verify the cloning by demonstrating the presence of insert in the plasmid. For this verification, there are several strategies:

* **Restriction enzyme digestion** (i.e. novel restriction enzyme in the insert, size change in the linearized plasmid)
* **PCR** (a primer pair flanking multiple cloning site, one primer attaches to the insert and the other attaches to the backbone)
* **DNA sequencing**

You will verify the cloning via PCR amplification. Forward primer was designed to attach the insert
while the reverse primer attaches to the backbone. Amplification of a 213 bp-long amplicon indicates
the presence of insert on the vector.

![](https://static.igem.wiki/teams/4974/wiki/experiments/picture3.jpg)

Figure 3: Verification of Cloning via PCR Amplification.

verification\_fwd:5’ GTTAGCGATGATGGTAAAGC 3’

verification\_rev: 5’ CCTCAAGACCCGTTTAGAG 3’

You can also verify successful insertion nucleotide by nucleotide via Next Generation Sequencing (NGS) to track point mutations if necessary.

## 8. Verification of Transformation (Colony PCR)

You can directly use colony plaques in PCR to verify the presence of plasmid in the clones. You should
prepare the PCR master mix by adding the primers above (verification\_fwd & verification\_rev). You
should first choose and mark the colonies (You will need the successful clones later). You should
transfer cells from each colony to a PCR tube using a sterile micropipette tip or a sterile toothpick
and briefly stir to resuspend them in the PCR master mix. Expected band size is ~200 bps.

## 9. Transformation into BL21

You should transform verified plasmid into the bacterial strain named BL21 for protein expression.
After transformation, successful clones should be grown in LB overnight. Please do not forget to take
cell stocks from the culture to have backups of the clones.

## Our Findings

As our team is fighting to lower the cost of medicine, we will utilize an expressed, synthetic alpha amylase protein from the BL21 cells, and then we can detect them by both western blot and aptamers. The western blot is the basis of an ELISA, a tried and true method. This would be our fallback to detecting our synthetic protein. Our preference is to build a new diagnostic device where synthetic aptamers containing a 5' fluorescent tag become bound to AA protein ([**Minagawa et al., 2017**](https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5335659/)). We then used a plate reader and emission spectra to determine bound vs unbound/free alpha amylase ([**Weaver and Whelan, 2020**](https://pubs.rsc.org/en/content/articlelanding/2021/ay/d0ay02256j)). The more bound signal, the more aptamer-protein complexes, the larger the fluorescent signal. Should this high signal persist over time with multiple buccal swabs, this is the points we would collect to determine when patients are experiencing acute stress and we would have a novel biomarker for acute stress.
Our negative control for the aptamer experiments would have been thrombin and DNA strands specific to thrombin ([**Liu et al., 2020**](https://jbioleng.biomedcentral.com/articles/10.1186/s13036-019-0223-y)). A second method to detect the alpha-amylase-aptamer complex would have been
[**through use of an EMSA**](https://www.thermofisher.com/us/en/home/life-science/protein-biology/protein-biology-learning-center/protein-biology-resource-library/pierce-protein-methods/gel-shift-assays-emsa.html#1) where the binding of the probe greatly reduces the migration of the protein through an acrylamide gel. We could have expanded upon the use of an aptamer based approach and included cortisol as well ([**Strahler et al., 2017**](https://pubmed.ncbi.nlm.nih.gov/28864234/)).

The use of an ELISA, fluorescent aptamers and an EMSA would have been three methods to prove our ability to detect an acute stress biomarker with alpha amylase for our novel diagnostic device. As our iGEM wiki freezes on October 12th, **we will continue to develop our aptamer research to present conclusive research in Paris.**

## ![](https://static.igem.wiki/teams/4974/wiki/icons/notepad-2-xxl.png) References

Minagawa, Hirotaka et al. “Selection, Characterization and Application of Artificial DNA Aptamer Containing Appended Bases with Sub-nanomolar Affinity for a Salivary Biomarker.” Scientific reports vol. 7 42716. 3 Mar. 2017, doi:10.1038/srep42716

Weaver, Simon D., and Rebecca J. Whelan. “Characterization of DNA Aptamer–Protein Binding Using Fluorescence Anisotropy Assays in Low-Volume, High-Efficiency Plates.” Analytical Methods, The Royal Society of Chemistry, 3 Feb. 2021, pubs.rsc.org/en/content/articlelanding/2021/ay/d0ay02256j.

“Gel Shift Assays (EMSA): Thermo Fisher Scientific - US.” Gel Shift Assays (EMSA) | Thermo Fisher Scientific - US, www.thermofisher.com/us/en/home/life-science/protein-biology/protein-biology-learning-center/protein-biology-resource-library/pierce-protein-methods/gel-shift-assays-emsa.html#1. Accessed 12 Oct. 2023.

Strahler, Jana et al. “Simultaneous measurement of salivary cortisol and alpha-amylase: Application and recommendations.” Neuroscience and biobehavioral reviews vol. 83 (2017): 657-677. doi:10.1016/j.neubiorev.2017.08.015

Liu, Qingxiu, et al. “Selex Tool: A Novel and Convenient Gel-Based Diffusion Method for Monitoring of Aptamer-Target Binding - Journal of Biological Engineering.” BioMed Central, BioMed Central, 13 Jan. 2020, jbioleng.biomedcentral.com/articles/10.1186/s13036-019-0223-y.

{% endblock %}

{% extends "layout.html" %}
{% block title %}Results{% endblock %}
{% block lead %}You can describe the results of your project and your future plans here.{% endblock %}
{% block page\_content %}

1. [PCR of Gene-of-Interest](#results1)
2. [Generation of Plasmid Backbone](#results2)
3. [Sequencing Using Plasmidsaurus](#results3)

## PCR of Gene-of-Interest

We made more of the human alpha-amylase gene insert (obtained from Integrated DNA Technologies) through PCR amplification.
The human alpha-amylase gene insert (GOI) appeared to be in the right location–between 1000bp-1500bp in length.
Between 1kb and 2kb of the PCR products ran clean because of the water.

![plasmid backbone](https://static.igem.wiki/teams/4974/wiki/results/results21.jpeg)

Figure 1: 07-25-23 PCR of GOI:

## Generation of Plasmid Backbone

We saw that the plasmid aligns with the 5kb mark of the DNA ladder. All six wells were 5kb in length, which was the result expected based on the sequence length of the plasmid backbone. However there was no clear indication that the 650bp product of the excised sequence of the plasmid backbone was present.
Although there are faint DNA bands along the 500 bp mark of the DNA ladder, they are most likely ghost bands“: artefactual bands frequently occurring, especially when partially homologous nucleic acids, such as splicing variants of DNA transcripts, are analyzed simultaneously.”

![plasmid backbone](https://static.igem.wiki/teams/4974/wiki/results/results22.png)

Figure 2: 07-28-23 Restriction Digest of plasmid for 60 min at 42C

The results for the gel electrophoresis of the restriction digest of our PET-28a plasmid appear at 5kb at 37C for 15 min and 60 min. We chose to test whether time was an issue because the gel results from our previous restriction digests were incoherent with the expected placement of the DNA bands in the DNA ladder. The protocol that we followed recommended we leave the restriction enzyme + plasmid backbone assay for 15 minutes. We hypothesized that maybe we didn’t give the restriction enzymes enough time to excise the DNA.
Our hypothesis was proven wrong and based on these results, Time is NOT the issue; the plasmid moves the same length regardless of length of time kept at 37ºC.
The 650 bp product is still not in the gel electrophoresis, making it clear to us that our first attempt at restriction digest was not a fluke or an anomaly.

![plasmid backbone](https://static.igem.wiki/teams/4974/wiki/results/results23.png)

Figure 3: 08-02-23 Restriction digest of plasmid for 15 min or 60 min at 37C

We tried increasing the concentration of the plasmid backbone in the assay with the two restriction enzymes to see whether there was not enough of the plasmid for the restriction enzymes to efficiently cut.
The DNA bands for the plasmid appear at 5kb at 37C for both 15min and 60min, further confirming our assumptions that time didn’t affect the restriction enzymes’ ability to cut the plasmid backbone.
We also learned that concentration is NOT the issue because the DNA bands for the plasmid move the same length regardless of the concentration of restriction digest of the plasmid.
Both decreased/increased combinations of time and decreased/increased concentrations of plasmid backbone in the assay containing the backbone and restriction enzymes did not seem to result in the 650 bp product.

![plasmid backbone](https://static.igem.wiki/teams/4974/wiki/results/results24.png)

Figure 4: 08-09-23 Restriction digest of plasmid for 15min OR 60min at 37C with DNA gradient

We tried different combinations of the restriction enzymes with the plasmid backbone to test for three possible reasons for the MCS not being properly excised: the XbaI restriction enzyme wasn’t working, the XhoI restriction enzyme wasn’t working, or both of the restriction enzymes weren’t working.
Just like the other gels, the Plasmid appears at 5kb at 37C when run through a thermocycler for 15 minutes and for 60 minutes.
The DNA bands for the assays with only one of either restriction enzymes clearly indicated that the enzymes were cutting the backbone as shown in the difference between the length of the uncut plasmid backbone and the individually cut plasmid backbones, but we didn’t see any 650 kb product.
We had several new hypotheses after this result: Enzymes are nicking the plasmid BUT not cutting the plasmid? Are the restriction sites mutated, not allowing the enzymes to cut? We sent out our plasmid for sequencing to help answer these questions.

![plasmid backbone](https://static.igem.wiki/teams/4974/wiki/results/results25.png)

Figure 5: 08-09-23 Restriction digest of plasmid for 30min at 37C with 1μg DNA

We ran a PCR on the multiple clonal site to determine if that was the cause of our error. The MCS appears to be around 0.7 kb long, which is close to the expected length of our excised sequence (0.65 kb).
We came up with possible reasons for why the restriction enzymes weren’t working after determining that the PCR of the flanking regions of the MCS were in fact around 0.65 kb long. There was a chance that we might have run the gel for too long. There was also a possibility that there was not enough of the plasmid backbone for the restriction enzymes to sufficiently locate and cut the target sequence. The volume of samples inserted into the well could have also been insufficient for the DNA to run completely in the gel.

![plasmid backbone](https://static.igem.wiki/teams/4974/wiki/results/results26.png)

Figure 6: 08-11-2023 PCR to the flanking regions of the MCS (Multiple Cloning Site)

At one point, we decided to start from scratch and isolate the PET plasmid DNA using a completely new mini-prep kit, as we realized the possibility our mini-prep kit was malfunctioning.
We measured 44.483 ng of plasmid DNA per uL \* 50 uL of plasmid DNA solution \* 6 samples ~ 13.2 ug of DNA.
This isolation of PET plasmid DNA using the new kit led to more of the plasmid being isolated compared to when the previous QIAgen miniprep kit. We decided to use this plasmid for mini-prep going forward.

![plasmid backbone](https://static.igem.wiki/teams/4974/wiki/results/results27.png)

Figure 7: 09-04-23, Isolation of PET plasmid DNA using new mini prep kit

Although we reordered a new plasmid (to see if the plasmid itself was the problem), the results showed that the restriction enzymes were still not cutting plasmid. This led us to consider future steps for us to consider in resolving this issue. We thought about ordering new enzymes because the restriction enzymes themselves might have been inaccurately chosen for our specific plasmid. We also considered the possibility that the plasmid is not what we think it is. In other words, either the design of the plasmid on benchling was inaccurate, leading to further errors in choosing the suitable restriction enzymes.

![plasmid backbone](https://static.igem.wiki/teams/4974/wiki/results/results28.png)

Figure 8: 09-07-23, Restriction digest of NEW plasmid for 30min at 37C with 1ug DNA following results from 08-11-23

We tested the new restriction enzymes that we ordered on the new overnight culture, changing two potential variables that could have explained the failure of our previous trials. We also used the highest resolution gel to date.
The results still showed missing excised MCS, meaning none of the variables/conditions listed above were issues.
A possible pivot we considered was to move to utilizing either a western approach (technique used to separate a mixture of proteins based on weight using gel electrophoresis) or an aptamer approach, to begin testing the viability of aptamer-based detection. We started to develop a new research plan for October to move past our restriction digest challenges to start aptamer testing…

![plasmid backbone](https://static.igem.wiki/teams/4974/wiki/results/results29.png)

Figure 9: 09.26.2023, Restriction digest with new enzymes

## Sequencing Using Plasmidsaurus

After receiving these results, we felt that we had no other option but to ship in our samples for sequencing to see if our problem was due to an error in the sequence itself. We attempted to send our samples to Plasmidsaurus. However, the company notified us that they were not able to obtain any results, likely do to nuclease degradation.

![email](https://static.igem.wiki/teams/4974/wiki/results/results2.jpg)

Figure 10: An email from Plasmidsaurus explaining that our plasmid sequencing did not work.

{% endblock %}