iGEM 2022 — Team east-coast-biocrew

## Lab Notebook

### Genspace Lab Notes

### 5/30/2022

#### Reporter Options:

* beta-galactose (colorimetric)
* luciferase (light)
* RFP (fluorescence)
* pH reporter assay
* ELISA (colorimetric)

If not our microplate reader, how will we detect color change?

Magnetic bacteria: [*We need to domesticate more microbes*](https://www.microbyre.com/blog/domesticate-microbes/)

#### Homework:

* Casey to look through 3 previous iGEM projects
* Everyone to search/be ready to describe 2-3 options for reporter assays
* Beth to ask Chaz to look into Arduino/Raspberry Pi sensor options

#### Notes on labwork:

Goal: get a feel for 3A cloning/assembly

* \*biobricks (see 3A Assembly Protocol)
* Goal: get a feel for 3A cloning/assembly
* Standard set of restriction enzymes --> swap out parts like legos
* Used Pst I and EcoRI
* Anything under an iGEM project should be 3A assembly compatible
* Everything has the same "standard connection" from an engineering point of view
* You can stitch things together at different restriction sites

In our plasmid we have PstI and EcoRI on either side of the insert (RFP or GFP) --> flanked by the restriction enzymes

Depending on which 3A backbone plasmid (different antibiotic resistance) --> have to be outside of the section GFP/RFP flanked by the Pst1 and EcoR1:

* Amp
* Kan
* Chlor

#### Cut & digest with PstI and EcoRI:

GFP/RFP (writing for GFP but same idea)

Physically cutting out the GFP but the pSB1A3 (AmpR) backbone is the same in both GFP and RFP --> we ligate the
opposite GFP and RFP into the opposite backbones --> it works because the EcoR1 and Pst1 will still attach at
the same sites --> all will grow on Amp plates

Ligation means to "glue back together"

Last step: transformation --> transfer the new plasmids into E. coli onto Amp plates

Those that have succesfuly been transferred into E. coli survive / the E. coli without the plasmids transferred will die because they lack Amp resistance

### 7/1/2022

The start of getting the actual sequences for this paper: 10.1074/jbc.272.52.32824

Essentially this was an attempt to start rebuilding some of the coding regions that were talked about in that
paper, even though I'm not sure I know what I'm doing
The first thing I did was find the FASTAs for human AhR and ARNT, as well as a two way promoter that promotes
in both directions when exposed to galactose and is repressed by glucose. The paper then has this image:

![An image of three boxes, reading from left to right: ARNT, GAL 1 10 Promoter, AHR](https://static.igem.wiki/teams/4458/wiki/gr1.jpg)

And so took the reverse compliment of the ARNT dna and stuck it all together end to end, which is the ARNT rev
comp> GAL 1,10 2way promoter > AhR sequence. Unless there's something I'm missing, in theory this base sequence
should directly code for the genes in the diagram.

This is all I had time for at this current time due to my program ending and a flurry of final project, but now
that I will be going back to New York soon I will be dedicating a lot more time to this project and should have
draft plasmids ready to go fairly quickly.

### 8/13/2022

Notes from BT re: Gibson Assembly

[Blog Post](https://blog.addgene.org/plasmids-101-gibson-assembly) - takeaway messages:

* Recommends 15-40bp overlaps with melting temp > 48C
* Look out for secondary structure in the overhang sequence
* Works best with fragments > 200bp (exonuclease may digest fragment before it anneals/polymerizes)

### 8/30/2022

* Measured and mixed 8g LB agar and 200 mL and autoclaved at 121 C for 20 min.
* After autoclaving, added 200 uL of 50 mg/mL Ampicillin Stock(1000x)
* Poured 15-20 mL per plate
* Dried the plates for 30-35 minutes
* Streaked tonight

#### Yeast Plasmids Parts

* AHR / ARNT sensor (13kb) with URA3 selection marker
* CYPC1A / Luciferase reporter (11kb) with LEU2 selection marker (CYPC1A is eukaroyote promoter)
* Backbone BF303B
* Gene inserts Twist (6 pieces, each piece 1800 bp) for 5.8kb

Use Gibson assembly to stitch backbone and Twist inserts to make PLK001

#### Notes

* ori for bacteria and 2ori for yeast
* Grow plasmids in bacteria to make more plasmids

#### Things to Validate/Test

* Response of CYPC1A to AHR / ARNT
* Quantity of luciferase to PCB

### 8/31/2022

* Transferred 20mL of LB Broth to a 50 mL conical tube in the sterile hood
* Added 20 uL of 50 mg/mL Ampicillin Stock (1000x) to the conical tube in the sterile hood
* Transferred 3 mL each of LB + Amp into six 15 mL conical tubes
* Transferred single colonies from plates grown overnight to tubes
* Three tubes were inoculated with a single colony each of pBF3038
* Three tubes were incolulated with a single colony each of pIGAhRC
* Rotated at 37 C overnight

### 9/1/2022

#### MiniPrep

* Followed MiniPrep according to Genspace Protocol(https://benchling.com/s/prt-jvYx7x8SrS9lZZC9TITZ?m=slm-LFfwpQR2WMXVxY4j3DaJ), started with 1.5 mL of culture.
* On the last step, eluted with 30 uL Elute Buffer, centrifuged 1 min., picked up flow through, reapplied flow through the column, and centrifuged for 1 min.

#### QuBit

* Followed [Genspace Protocol](https://benchling.com/s/prt-krVZlD6784pKdJ29HRo6?m=slm-VPeDJdGsdan633YoKcuU)
* Used 2 uL of plasmid DNA Sample and 198 uL Mastermix

#### Results:

|  |  |  |  |
| --- | --- | --- | --- |
|  | Luke | Naomi | Maya |
| pBF3038 | 157 ug/mL | 192 ug/mL | 184 ug/mL |
| pIGAhRC | 58.7 ug/mL | 68.9 ug/mL | 64.5 ug/mL |

#### Glycerol Stock:

* Made a 30% Glycerol Stock
* Used 500 mL of 60% Autoclaved Glycerol and 500 mL of Culture
* Stored -80C Freezer

#### Reconstituting Primers:

* Took brand new primers, labeled them with shorthand names, and diluted them to 100 uM in the original container by adding 10x of their molecular weight in sterile water
* Made a working stock(10 uM) by adding 90 uL of sterile water, 10 uL of diluted sample, vortexed.
* Stored in -20C FreezerTher

### 9/7/2022

#### Day 1: Bacterial Transformation

* Take SOC media out of the fridge to warm to Room Temp (use the one that's labeled expired 6/21, not Open Plant's bottle) - take a 650ul aliquot out in the sterile hood
* Take 3 LB+Amp plates out of the fridge to warm to Room Temp
* Thaw 4 aliquots of cells on ice
  + **Gibson:** Add 5ul of chilled assembly product to competent cells (mix gently by flicking, do not vortex)
  + **Positive Control:** Add 1ul of the pBF3038 diluted 1:10 in water
  + **Positive Non-Diluted Control:** Add 1ul of the pBF3038
  + **Negative Control:** no DNA
* Place the mixture on ice 30 min
* Heat shock @42C for 30 seconds
* Place mixture back on ice for 5 min
* Add 200 ul of SOC to the tube
* Add 250 ul of cells + media to the LB+Amp plates - spread using the glass beads
* Incubate O/N @37C

### 9/8/2022

#### Day 2: Check Results + Start O/N cultures for Miniprep

Take plates out of the incubator (AM), store at 4C until ready to use

At ~5-8pm

* Take LB out of the fridge, aliquot 3ml each for 2-6 clones (depends on how efficient the transformation is) in 15ml Conical tubes + Amp
* so for 2 clones, 6ml LB + 6ul Amp --> mix and split into 2 tubes
* 6 clones = 18ml LB + 18ul Amp --> mix and split into 6 tubes
* Make sure lids are on tight, and place in the end-over-end rotator inside the 37C incubator (turn on the rotator)
* Grow O/N

### 9/9/2022

#### Day 3: Miniprep & Qubit

Follow Miniprep Protocol and [Qubit](https://benchling.com/lscheifele/f/lib_hGSfg4ch-in-person-lab-notebook/prt_dB3keNlU-genspace-qubit-protocol/edit) as before

* Miniprep Protocol followed exactly (added 30 ul of Elution Buffer)

+ 4 minipreps were created with the four colonies that were rotated overnight

* 4 Qubit samples from the 4 minipreps

Used 3 ml of overnight rotations in the miniprep

|  |  |  |  |
| --- | --- | --- | --- |
| G1 | G2 | G3 | G4 |
| 34.3 ug/ml | 16.4 ug/ml | 20.8 ug/ml | 8.95 ug/ml |

Qubit samples were slightly vortexed to ensure accurate results. Qubit was recalibrated

|  |  |  |  |
| --- | --- | --- | --- |
| Sample 1 | Sample 2 | Sample 3 | Sample 4 |
| 21.9 ug/ml | 10.9 ug/ml | 26.1 ug/ml | 11.0 ug/ml |

#### Day TBD: Sequencing

Genspace Protocol (+ get some assistance from Jehovani or arrange a Zoom call with BT to complete these steps)

#### Modifications to the standard PCR product ordering:

#### Sample Prep

* Prep Samples for Sequencing with same labeling: 1 on the lid, AA01 on the side, etc. in strip cap PCR tubes
* + Aim for 10-15ul at 50ng/ul
* Prep Primers in 500uL total volume at 5uM in 1.5ml tubes (25ul of 100uM stocks + 475 fancy sterile nuclease-free water)

#### Azenta Website (ask Jehovani/BT for help)

* Select Plasmid
* Select Predefined (may use premixed depending on the number of clones you want to sequence)
* Select Standard Priority
* Enter the # of samples
* Order Name: iGEM2022 pLK001 Clones
* DNA Name: pLK001\_# of the clone
* Length: <500bp
* Concentration: Include from Qubit (and/or standardize concentrations to 50ng/ul) and
* Primer Name: Include all primers: 1F;1R;2F;2R;3F;3R;4F;4R;5F;5R;6F;6R;7F;7R
* Submit

Shipping/FedEx - follow the protocol (ask Jehovani/BT for help)

#### Day TBD: Yeast Transformation

Making Media: Yeast SDO Plates -Ura AND -Leu

[Genspace Media Protocol](https://docs.google.com/document/d/1yFoEjCEoveJrmrB47wW6fEcQcCHWZo7cxA4PEGiGmSg/edit), adapted recipe:

* 20g/L Bacteriological Agar
* 6.7g/L Yeast Nitrogen Base
* 1.62g/L Yeast Synthetic Drop-out Medium -His, -Leu, -Trp, -Ura
* 0.4g/L L-Tryptophan
* 0.2g/L L-Histidine

+ make Dextrose/glucose spearately - recombine after autoclaving, before pouring plates (as with other YPD Agar)

\*Also make a batch with no agar for liquid cultures

[Yeast Transformation](https://files.zymoresearch.com/protocols/_t2001_frozen-ez_yeast_transformation_ii.pdf)

\*Grow fresh cells from BY4741 stocks

Select transformants to work with - make frozen glycerol stocks

Assay Function

Restriction digest of pBF3038 to remove the 1500bp sequence between the f1 ori and CYC1 Terminator (make the plasmid compatible with the Gibson Assembly Twist fragments)

* XbaI (CYC1)
* SpeI-HF (f1 ori)

[NobCloner](https://nebcloner.neb.com/#!/protocol/re/double/XbaI,SpeI-HF)

|  |  |
| --- | --- |
| Reagent | 25uL Rxn |
| 500 ng DNA | 2.5 |
| 10x CutSmart | 2.5 |
| XbaI | 0.5 |
| SpeI-HF | 0.5 |
| Nuclease-free H2O | 19 |
|  | 25 |

* Digest at 37C for 15 min
* Run 0.7% agaroe gel, 1x TAE, 2.5ul EtBR
* Run gel for ~30 min @120V

![An image showing the orange lines on a gel plate scan](https://static.igem.wiki/teams/4458/wiki/picture17.png)

5500 bp fragment

[Gel Extraction Protocol](https://www.neb.com/protocols/2015/11/23/monarch-dna-gel-extraction-kit-protocol-t1020)

* 336mg gel + 4x vol (1344ul) melt @ 52C for ~10 min
* Elute in 7ul nuclease-free water

[Qubit Protocol](https://benchling.com/lscheifele/f/lib_hGSfg4ch-in-person-lab-notebook/prt_dB3keNlU-genspace-qubit-protocol/edit)

* Digested, purified pBF3038 - 8.8ng/ul
* Proceed to Gibson Assembly

#### Gibson Assembly

* Digested pBF3038 to 1500bp remove fragment and make space for our Gibson construct
* Conducted gel electrophoriesis to seperate plasmid fragments and purify product
* Qubit(https://benchling.com/s/prt-krVZlD6784pKdJ29HRo6?m=slm-VPeDJdGsdan633YoKcuU) to measure DNA Concentration (8.8ng/ul)

|  |  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- | --- |
|  | Weight (ng) | Vol to 10ng/ul | Length (bp) | pmol | Volume (ul) | total pmol for Gibson |
| CYP1A1\_1 | 765 | 76.5 | 1149 | 0.0133895695 | 1.3 | 0.0174064404 |
| CYP1A1\_2 | 588 | 58.8 | 1142 | 0.0134716422 | 1.3 | 0.0175131349 |
| CYP1A1\_3.1 | 757 | 75.7 | 871 | 0.0176631635 | 1.1 | 0.0194294798 |
| CYP1A1\_3.2 | 828 | 82.8 | 599 | 0.025683832 | 1 | 0.025683832 |
| CYC1 | 675 | 67.5 | 377 | 0.0408079984 | 0.7 | 0.0285655989 |
| Akaluc CDS | 847 | 84.7 | 1705 | 0.0090232348 | 1.9 | 0.0171441462 |
| pBF3038 |  | 5 | 5500 | 0.0024615385 | 5 | 0.0123076923 |
| TOTAL |  |  |  |  | 12.3 | 0.1380503244 |

#### Gibson Assembly Protocol

|  |  |
| --- | --- |
| Component | 7 Fragment Assembly Vol (ul) |
| CYP1A1\_1 | 1.3 |
| CYP1A1\_2 | 1.3 |
| CYP1A1\_3.1 | 1.1 |
| CYP1A1\_3.2 | 1 |
| CYC1 | 0.7 |
| Akaluc CDS | 1.9 |
| pBF3038 | 5 |
| Gibson 2x MM | 25 |
| Nuclease-free H2O | 12.7 |
| Total Volume | 50 |

#### Notes:

* Due to low concentration of digested plasmid, we opted to go for ~equimolar ratios vs. total pmol
* Due to >6 fragments assembled, we opted for 25ul instead of the recommended 10ul 2x Gibson Master Mix
* We also let the reaction go for 90 min (instead of 60)
* BT went ahead and did a transformation on 9/9 just to test... (may need to order backup supplies and that will take some time to ship)
* Rest of the Gibson Assembly product stored at -20C until the team can continue with the transformation

### 9/16/2022

#### BOOMS

* 1X TAE running low -> made from 20mL of 50X TAE + 980mL dH2O
* Spe-HF running low -> spun it down in centrifuge
* Split 1 digest sample into 2 gel wells -> extraction results in low quatity (2ng/ul)
* Qubit mixed up BR and HS standards -> redid standards while plasmids waited for a bit -> inaccurate results (measured the second time after some time and concentration was drastically different, e.g. Luke's sample was originally 2.08ng/ul and became >20ng/uL to next few times measured after some time elapsed)
* Autoclave LB agar used parafilm (melting) + Amp resistance left out a bit longer before adding to LB agar
* Negative control plates used dirty beads -> redid only the steps for heat shock

1. ##### Make Gel

   Use 6-well comb, make 0.7% agarose gel (50uL TAE + 0.35g agarose), 1x TAE, 2.5ul EtBR
2. ##### Disgest Plasmid pBF3038

   Add the following reagent into a PCR tube:

   |  |  |  |  |
   | --- | --- | --- | --- |
   |  | Luke | Naomi | Maya |
   | pBF3038 | 157 ug/mL | 192 ug/mL | 184 ug/mL |
   | pIGAhRC | 58.7 ug/mL | 68.9 ug/mL | 64.5 ug/mL |

   1000ng = 1ug, 184ug per 1000uL = 184ng/uL

   |  |  |  |
   | --- | --- | --- |
   | Reagent | 50uL Rxn | 50uL Rxn |
   | 1000 ng DNA | 5.4347826087 | 5.4 |
   | 10x CutSmart | 5 | 5 |
   | XbaI | 1 | 1 |
   | SpeI-HF | 1 | 1 |
   | Nuclease-free H2O | 37.5652173913 | 37.6 |
   | Total Volume | 50 | 50 |

   Digest at 37C for 15 min
3. ##### Make Media for Plating

   Measured and mixed 8g LB agar and 200 mL of distilled water and autoclaved at 121 C for 20 min (3rd conical
   flask settings). After autoclaving, added 200 uL of 50 mg/mL Ampicillin Stock (1000x). Poured 15-20 mL per
   plate. Dried the plates for 30-35 minutes.
4. ##### Run Gel

   * 15uL of 100kb extended DNA ladder
   * Add 10uL of dye into each 50uL sample (1:6X loading dye)
   * Load 50uL into 2 wells each (should give 4 times as much)
   * Load very slowly, will spill if too fast
   * Run gel for ~30 min @120V
   * Loaded into 3 wells
5. ##### Gel Extraction

   Follow [protocol](https://www.neb.com/protocols/2015/11/23/monarch-dna-gel-extraction-kit-protocol-t1020) from Monarch DNA Gel Extraction Kit

   * Tube 1: 0.19g ; Tube 2: 0.39g (split tube into 2 smaller ones)
   * Tube 1: 190mg (760ul)
   * Tube 2A: 0.182g (728ul)
   * Tube 2B: 0.211g (844ul)
   * use 2 spin columns
   * 8uL of elution buffer
   * combine the 2 spin column volumes (2uL for Qubit) => 12uL
   * reload elution buffer for a second spin
6. ##### Qubit

   Aim for concentration of digested, purified pBF3038 > 20ng/ul (5uL = 100ng)

   * Tube 1: 20.1 ug/ml
   * Tube 2: 2.08ug/ml
7. ##### Gibson Assembly

   [Protocol](https://www.neb.com/-/media/nebus/files/manuals/manuale2611_e5510.pdf?rev=c2a9c78a94af4ee29da34b62cf4ddef2&hash=AD2431F95B100290CBFBEEDB6AB0F807) reference from NEB

   * 50–100 ng of vector with 2-3 fold molar excess of each insert
   * Can aim for 200ng of vector
   * pmol = (concentration\*1000)/(D8\*650)
   * Available stock: 200uL 2X Gibson MM
   * Do 2-3 Gibson reactions to test:
   * Tweak pmols of inserts
   * Tweak more Gibson MM or total volume to make sure insert fragments less than 20% (~40uL)
   * Incubate samples in a thermocycler at 50°C for 90 minutes
   * Store samples on ice or at –20°C for subsequent transformation.

   |  |  |  |  |  |  |  |
   | --- | --- | --- | --- | --- | --- | --- |
   |  | Weight (ng) | Vol to 10ng/ul | Length (bp) | pmol | Volume (ul) | total pmol for Gibson |
   | CYP1A1\_1 | 765 | 76.5 | 1149 | 0.0133895695 | 2.1 | 0.028118096 |
   | CYP1A1\_2 | 588 | 58.8 | 1142 | 0.0134716422 | 2.1 | 0.0282904486 |
   | CYP1A1\_3.1 | 757 | 75.7 | 871 | 0.0176631635 | 1.7 | 0.0300273779 |
   | CYP1A1\_3.2 | 828 | 82.8 | 599 | 0.025683832 | 1.2 | 0.0308205984 |
   | CYC1 | 675 | 67.5 | 377 | 0.0408079984 | 0.8 | 0.0326463987 |
   | Akaluc CDS | 847 | 84.7 | 1705 | 0.0090232348 | 3.2 | 0.0288743515 |
   | pBF3038 |  | 6 | 5500 | 0.0056223776 | 4.9 | 0.0275496503 |
   | TOTAL |  |  |  |  | 16 | 0.2063269214 |

   |  |  |  |
   | --- | --- | --- |
   | Component | 7 Fragment Assembly Vol (ul) |  |
   | CYP1A1\_1 | 2.1 |  |
   | CYP1A1\_2 | 2.1 |  |
   | CYP1A1\_3.1 | 1.7 |  |
   | CYP1A1\_3.2 | 1.2 |  |
   | CYC1 | 0.8 |  |
   | Akaluc CDS | 3.2 |  |
   | pBF3038 | 4.9 |  |
   | Gibson 2x MM | 17.5 | half of total volume |
   | Nuclease-free H2O | 1.5 |  |
   |  | 35 |  |
8. ##### Plating

   * Take SOC media out of the fridge to warm to Room Temp (use the one that's labeled expired 6/21, not Open Plant's bottle) - take a 650ul aliquot out in the sterile hood
   * Take 3 LB+Amp plates out of the fridge to warm to Room Temp
   * Thaw 3 aliquots of cells on ice
   + **Gibson:** Add 5ul of chilled assembly product to competent cells (mix gently by flicking, do not vortex)
   + **Positive Control:** Add 1ul of the pBF3038 diluted 1:10 in water
   + **Negative Control:** no DNA* Place the mixture on ice 30 min
   * Heat shock @42C for 30 seconds
   * Place mixture back on ice for 5 min
   * Add 200 ul of SOC to the tube
   * Add 250 ul of cells + media to the LB+Amp plates - spread using the glass beads
   + **Negative:** Just competent cells
   + **Positive:** pBF dilution* Incubate O/N @37C

### 10/1/2022

#### Materials:

* 4 Erlenmeyer flasks
* Weigh boats
* G and ml scale

#### Yeast Inoculation:

* Poured 11.5 mL of YPD Broth into a conical tube with approximatgely 50 uL of stock S. cerevisciae

+ Incubated in rotator at 30C for 5 hours --> BY 4741

* Custom Media for selecting for our plasmids:

+ Values in table obtained from [Sigma Aldrich](https://www.sigmaaldrich.com/deepweb/assets/sigmaaldrich/product/documents/169/136/y2001dat.pdf)

* **Started with:**
* SDO --> syntetic droupout media (SDO-4)

+ his
+ trp
+ leu
+ ura

* Histodine and Tryptophan get added back to end up the desired missing components

+ his
+ trp
+ leu
+ ura

We need 10 plates with 20 mL per plate so 20 mL per plate x 10 plates = 200 mL (for Agar)

Sigma Aldrich's concentrations are in mg/L or g/L, so we took 1/5 of all of those values to find out how much we needed, displayed in the table below.

|  |  |  |  |  |  |  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| Flask 1 | Concentration (mg/L) |  | Flask 2 | Concentration (mg/L) |  | Flask 3 | Concentration (g/L) |  | Flask 4 | Concentration (g/L) |  |
| Trp | 76 | 15.2 | Trp | 76 | 15.2 | Dextrose | 20 | 4 | Dextrose | 20 | 4 |
| His | 76 | 15.2 | His | 76 | 15.2 | Bact Agar | 20 | 4 | YNB | 6.7 | 1.34 |
| SDO-4 | 1390 | 278 | SDO-4 | 1390 | 278 |  |  |  |  |  |  |

Flasks were autoclaved at 121 degrees C / 26 minutes after each component was added to the flasks, listed above

Continuously quantified the Optical Density of the Yeast inoculation as it was in a rotator

* Added 1 mL of growing yeast culture and empty media to cuvettes and compared their optical densities
* 0.02 around 11:00
* 0.1 around 2:30
* 0.18 around 4:30

Poured plates, only added dextrose to plates since the liquid media should be kept seperate of galactose (in agarose) or glucose so as not to disrupt the GAL1,10 promoter. Plates will have both galactose and glucose, so they should cancel each other out (hopefully).

**Flask 5:**

* Created because dual expression vector does not express if there is glucose but expresses if there is galactose --> this flask contains a concentration of galactose ONLY --> for the controls
* 0.016% D-Galactose g/100 ml --> 0.032 g/200 ml (making 200 ml total in flask)
* still adding 32 mg to 100 ml water because we will add flask 5 into flask 2 --> need ratios to be the same (ratios of things we put in them to be the same) --> might not add flasks in entireties
* Autoclaved flask 5 for 26 minutes at 121 degrees C

### BUGSS Team Lab Notes

### 5/21/2022

#### Notes For The Bacterial Degredation of Polychlorinated Benyphyls

* This procedure is taking genes from prokaryotic bacteria to create eukaryotic yeast
* The goal is to combine a total of 9 genes and insert them into 5 plasmids from 4 different bacteria into yeast
* The previous team was only able to do 1 or 2 genes successfully
* BphAI+2 was one of the genes that successfully assembled as DNA
* Only the BPHA1 protein was able to be verified (opperating correctly and forming mRNA). It was verified with Western blot using Flag-tag.
* The PCBA5 enzyme was successfully applied to transfer the PCBs from highly chlorinated to less chlorinated
* Five enzymes are needed to be applied to make the PCBs n-chlorobenzoate with 2-hydroxy pentanoate
* Three enzymes are then needed to be applied to make the benzoate pyruvate to acetyl coa

### 5/28/2022

* Measured concentration by Nanodrop
* Calculate volume of plasmid and amount of water needed to get 25 uL of each template

|  |  |  |  |  |
| --- | --- | --- | --- | --- |
| Template DNA | Concentration (ng/uL) | A260/280 | Volume of Plasmid (uL) | Amount of Water (uL) |
| RFP | 11.1 | 1.83 | 2.3 | 22.7 |
| Joy | 103.4 | 2.07 | 0.24 | 24.76 |
| AFP Plasmid | 16.7 | 1.85 | 1.5 | 23.5 |
| 11.2 Plasmid | 17.1 | 1.8 | 1.46 | 23.54 |

### 7/30/2022

Performed ligation and transformation of original PCBA5 gene. No successful bacteria colonies.

### 8/6/2022

Repeated transformation with 3 samples:

* Positive control (RFP; J04450 plasmid)
* PCBA5 gene
* Negative control (water)

Plated on LB chlor plates and incubated at 37 C

#### Transformation:

* Thaw 3 tubes 25uL competent cells on ice.

*\*Dry off the lid and label them Lig (1 for each student), RFP, GFP, and Ctrl (for Negative Controls)*

* Add 1uL of ligated DNA to Lig tube and mix gently by flicking the tube 4–5 times. Do not vortex. Add 1uL of 5 ng/ul diluted plasmid plasmid to the corresponding positive control tubes. No DNA into Ctrl tube.
* Place the mixtures on ice for 15 minutes. Do not mix.
* Heat shock at 42°C for 30 seconds. Do not mix.
* Place on ice for 5 minutes. Do not mix.
* Add 200 µl of room temperature LB media to the tube.
* Label the bottom of your LB Agar plates with the Sample ID, Your Initials, and Date
* Spread 150 µl of the cells and plasmid mixture onto the appropriate plates using the glass beads.

*\*Dispose of used beads in the dirty bead container (do not throw away)*

* Incubate overnight at 37°C.
* Analyze results during the next session.

### 8/20/2022

Resuspending, digesting, and ligating PCBA4, PCBA1, and PCBA5 with sticky-end revised to the vector PSB1c3.

|  |  |  |
| --- | --- | --- |
| PCBA1 | 24.4 ng/uL | 0.88 |
| PCBA4 | 29.2 ng/uL | 0.80 |
| PCBA5 | 25.0 ng/uL | 0.96 |
| PSB1c3 | 21.4 ng/uL | 0.94 |

### 8/27/2022

* Transformed bacteria following protocol
* Plated bacteria

### 9/10/2022

* Attempt to culture bacteria from 8/27 (ie. with sticky-end linear vector) yeilded no successful colonies
* Set up following 10 uL ligation reaction with PCR products (PCBA1, PCBA4, PCBA5) in separate tubes:

|  |  |  |
| --- | --- | --- |
| Reagent | Volume | Final Concentration |
| 5x Ligase Buffer | 2 uL | 1x |
| Blunt-end linear vector | 1 uL | 20 ng/uL |
| PCR product | 1 uL | 30 ng/uL |
| Molecular Biology-Grade Water | 5 uL |  |
| T4 DNA Ligase | 1 uL | 200 U/uL |

* Incubated ligation reaction at 25 C

### 9/17/2022

* *Rhodocuccus* medium synthesis:

+ Stock salt solucion: Missing MnSO4, left out HCl from solution
+ Concentrated Goodies requires Stock Salf, which is incomplete as of 9/17
+ Mg/Ca?B1 goodies mix: everything except Concentrated Goodies mixed together and stored at 4 C
+ Basal Salt solution: complete

* Blunt-end ligation in *E. coli*

+ Transform bacteria following protocol (30 min before heat shock, 30 sec heat shock, hour incubation after SOC addition)
+ Plate bacteria in LB Growth plates
+ Check on growth next Saturday

### 9/24/2022

**Screening *E. coli* colonies**

9/17 bacteria (SmartJoin plasmid)

* Ligation negative control = 0 colonies
* Negative coltrol = 0 colonies
* Ligation positive control = 30 colonies
* PCBA1 = 1 colony
* PCBA4 = 1 colony
* PCBA5 = 12 colonies

8/27 bacteria (PSB1c3 plasmid)

* Negative = 0 colonies
* Positive = 3 pink, 6 white
* PCBA1 = 2 pink, TMTC white
* PCBA4 = 1 pink, TMTC white
* PCBA5 = 3 pink, 1 white
* PCBA1 Lig = 10 pink, 7 white
* PCBA4 Lig = 30 pink, 22 white
* PCBA5 Lig = 38 pink, 25 white

Performed colony PCR on the plated colonies

Performed gel electrophoresis on the amplified DNA for screening of PCBA1, PCBA4, and PCBA5

Completed synthesis of *Rhodocuccus* growth media, 100 mL total

### 10/1/2022

#### *Rhodococcus* Growth Media Procedure

* Create the stock salt solution by combining 22.94 g/L MgCl2-6H2O, 2.0 g/L CaCO3, 4.5 g/L FeSO4-7H2O, 1.44 g/L ZnSO4-7H2O, 0.85 g/L MnSO4-H2O, 0.25 g/L CuSO4 - 5H2O, 0.24 g/L CoCl2-6H2O, 0.06 g/L H3BO3, 51.3 mL HCl
* Create the concentrated goodies by combining 50 mL stock salt solution, 3.009 grams of MgSO4, 25 mL 1% FeSO4
* Bring the volume of the concetrated goodies up to 100 mL
* Sterilize by filtration and store in fridge
* Create 100X Mg/Ca/B1/Goodies mix by combining 60 mL 1M MgSO4, 3 mL 1M CaCl2, 3 mL 10 mM thiamine, and 75 mL concentrated goodies
* Adjust volume to 300 mL, sterilize by filtration and store at 4 degrees
* Create basal salts by combining 30 gram KH2PO4, 60 grams Na2HPO4, 5 grams NaCl, 10 grams NH4Cl
* Autoclave to serilize
* Create final growth media by combining 100 mL 20% dextrose, 10 mL Mg/Ca/Bl/Goodies, 200 mL basal salts, and 690 mL water

#### *Rhodococcus* Optical Density Procedure

* Measure optical density of starting cell cultures (*Rhodococcus*, *E. coli*)
* Calculate the total volume of the cell cultures c1 v1 = c2 V2
* Create *Rhodococcus* growth media by mixing 100ml 20% dextrose, 10ml Mg/Ca/Bl/Goodies, 200Ml basal salts, and 690 ml water
* Combine *Rhodococcus* and *Rhodococcus* growth media in a flask
* Combine *E. coli* and *Rhodococcus* growth media in a flask
* Combine *E. coli* and LB growth media in a flask
* Combine *Rhodococcus* and LB growth media in a flask
* Place all flasks into the incubator shaker and check the concentration of cells every half hour for 5 hours

|  |  |  |  |  |
| --- | --- | --- | --- | --- |
| Time | *Rhodocuccus* in Rhodocuccus media | *Rhodococcus* in LB media | *E. coli* in Rhodococcus media | *E. coli* in LB media |
| Initial | 0.306 | 0.306 | 0.630 | 0.630 |
| 30 mins | 0.064 | 0.099 | 0.138 | 0.172 |
| 60 mins | 0.063 | 0.087 | 0.150 | 0.214 |
| 90 mins | 0.065 | 0.080 | 0.166 | 0.276 |
| 120 mins | 0.064 | 0.065 | 0.190 | 0.394 |
| 150 mins | 0.064 | 0.210 | 0.067 | 0.631 |
| 180 mins | 0.120 | 0.103 | 0.238 | 0.891 |
| 210 mins | 0.085 | 0.109 | 0.344 | 1.149 |
| 240 mins | 0.081 | 0.098 | 0.481 | 1.500 |
| 270 mins | 0.102 | 0.113 | 0.634 | 1.731 |
| 300 mins | 0.106 | 0.094 | 0.776 | 1.856 |

### 10/8/2022

Repeating last week's experiment at 30 C instead of 37 C

|  |  |  |  |  |
| --- | --- | --- | --- | --- |
| Time | *Rhodocuccus* in Rhodocuccus media | *Rhodococcus* in LB media | *E. coli* in Rhodococcus media | *E. coli* in LB media |
| Initial | 0.191 | 0.191 | 2.850 | 2.850 |
| 30 mins | 0.054 | 0.086 | 0.093 | 0.117 |
| 60 mins | 0.039 | 0.063 | 0.048 | 0.096 |
| 90 mins | 0.067 | 0.066 | 0.131 | 0.104 |
| 120 mins | 0.055 | 0.073 | 0.051 | 0.138 |
| 150 mins | 0.060 | 0.090 | 0.058 | 0.220 |
| 180 mins | 0.077 | 0.081 | 0.072 | 0.267 |
| 210 mins | 0.074 | 0.135 | 0.088 | 0.330 |
| 240 mins | 0.068 | 0.072 | 0.115 | 0.425 |
| 270 mins | 0.104 | 0.101 | 0.123 | 0.484 |
| 300 mins | 0.185 | 0.126 | 0.109 | 0.570 |
| 350 mins | 0.171 | 0.180 | 0.140 | 0.721 |
| 380 mins | 0.170 | 0.200 | 0.123 | 0.763 |
| 410 mins | 0.187 | 0.217 | 0.128 | 0.851 |
| 470 mins | 0.256 | 0.327 | 0.137 | 1.127 |
| 520 mins | 0.364 | 0.432 | 0.156 | 1.303 |

## Engineering Success in the Development of the Degredation Pathway

#### Design

The concept of our project, to insert genes that are known to degrade Polychlorinated biphenyls into an organism,
was inspired by [Stockholm's 2020 project](https://2020.igem.org/Team%3AStockholm). In their project, they inserted their genes into yeast, but we were
unsure about yeast’s capabilities as a chassis for use in waterways. We researched other organisms to possibly use by reading
literature before settling on the *Rhodococcus* bacteria. *Rhodococcus*, while already displaying a
natural inclination to degrade PCBs, can also survive in aerobic and anaerobic environments, which would be
useful for treating PCB contamination in all environments. It has also been shown in literature that foreign
genes can be inserted into *Rhodococcus*.

A difficulty we identified in the engineering process was that the term PCBs describes a wide variety of chemical
congeners with differing amounts and positions of chlorines on phenyl rings. Commercial mixtures of PCBs, known in the United States as Aroclors, are complex mixtures of 60–90 types, congeners, of PCB molecules that differ in the number and position of chlorines on the phenyl rings. However, we found in literature that the genes PCBA1, PCBA4, and PCBA5 of the dehalogenese strains have been found to naturally degrade many different congeners of PCBs [1]. Each of the genes produce enzymes that target different chlorines in different positions and each targets dozens of the congeners making them an effective dechlorination force. In response to these findings, we decided to insert these three genes into our chassis of *Rhodococcus*.

![Diagram showing the dechlorination reactions which are caused by pcbA1, pcbA4, and pcbA5](https://static.igem.wiki/teams/4458/wiki/dechlorination2.png)

Dechlorination reactions caused by pcbA1, pcbA4, and pcbA5

We decided to insert these genes into *Rhodococcus* using pSB1C3 as a
vector. We thought this vector, being a standard vector for iGEM, would be a good place to start when engineering
since we could not find adequate sources for other *Rhodococcus* vectors.

#### Build

To build our plasmids we performed these steps:

1. Identify the genes in the article that explained the genomic characterization of the three unique *Dehalococcoides* [2]
2. Looked for the specific portion of the genome in Genbank
3. Exported the gene to Benchling
4. Performed codon optimization for *Rhodococcus* using the Genscript tool [3]
5. Made sure to remove restriction sites: Xbal, Pstl, Spel, EcoR1
6. Found a promoter used in the paper, An Integrative Toolbox for Synthetic Biology in
   Rhodococcus, PM6-OP3 [4]- Used a common RBS, BOO34
   - Used a common terminator that was also used by the 2016 Edinburgh team that worked on PCBs, [B0015](http://parts.igem.org/Part%3ABBa_B0015)

#### Test

Once we received the genes from IDT, we performed ligations and transformations with them. However, no successful colonies were created. We resuspended, ligated, and transformed the genes again. Once more, no successful colonies were created.

#### Iteration

We therefore redesigned our system so that instead of using pSB1C3, we used the SmartJoin vector which accepts any blunt DNA piece. Now when we resuspended, ligated, and transformed the genes into *E. coli* we got colonies. We screened them by colony PCR and got a band of the correct size, which shows that our engineering worked. We are preparing to transform the plasmids into *Rhodococcus* now to show that they work there also.

## Engineering Success in the Development of the Detection Pathway

#### Design

Present methods of PCB detection require long lengths of time; the real-time monitoring of sites must be accessible and efficient to minimize exposure throughout the food chain. Also, large efforts for the detection of PCBs occur only in largely contaminated sites in the status quo, leaving PCBs in areas with undetected quantities to pile up [5]. PCBs are difficult and expensive to measure because the composition of the PCBs changes over time, meaning that the relative level or amount of PCBs present in a certain space also changes over time. Since the composition changes, the toxicity can also change, making it hard to get a consistent and accurate reading. [6]

PCBs are most commonly detected using gas chromatography-mass spectrometry. This technique is considered the gold standard because it can explicitly identify organic molecules in complex mixtures. Gas chromatography-mass spectrometry needs a high vacuum, which requires maintenance, which is time consuming and expensive. It also requires organic solvent extraction to concentrate PCBs for its analysis, which can have additional environmental and health risks [7]. Limitations of these methods are that they are based on the availability of materials and facilities with gas chromatography columns or complex chemical analytical techniques. They determine the total concentration of pollutants, which overestimates the risk of impact on living organisms as only a fraction of the total amount of pollutant is the bioavailable fraction; the inability to distinguish between the two fractions of pollutants poses a major disadvantage to current, expensive, and time-consuming techniques.

The recent practice of biosensing by utilizing synthetic biology and the advantages of fusing reporter genes, such as lux, gfp, or lacZ, to responsive promoters and has begun to be used for PCB detection. In 1998, Layton et al. reported a bioluminescent reporter strain, Ralsonia eutropha ENV307 (pUTK6), detecting the bioavailability of PCBs through inserting the biphenyl promoter directly upstream of the bioluminescence genes [8]. All such biosensors are used in vitro and in environments such as soil.

We searched for a new, efficient, and accurate method of biosensing through taking advantage of the dioxin-responsive elements (DRE) of the Murine CYP1A1 gene promoter and the expression of the human aryl hydrocarbon receptor complex in yeast (AHR). Ultimately, luciferase will be produced and measured to detect PCBs quickly and efficiently.

![Cell containing both our PCB Sensor and PCB Responder, showing how the two will interact to product luciferase and emit light](https://static.igem.wiki/teams/4458/wiki/yeast-sensor.png)

Signalling pathway between our PCB Sensor and PCB Responderder

**Sensing**

To detect PCBs, we created a sensor containing:

* Bidirectional promoter between yeast GAL1 and GAL10 genes
* Promoter regulates expression of complex: AhR (Human Aryl Hydrocarbon Receptor) and ARNT (Aryl Hydrocarbon Receptor Nuclear Translocator)
* The complex translocates to nucleus when it binds PCBs

![Construct diagram showing a bidirectional GAL1,10 promoter regulating expression of AhR and ARNT](https://static.igem.wiki/teams/4458/wiki/sensing.png)

Construct design for our PCB Sensor

The human AHR complex and hydrocarbon receptor nuclear translocator protein (ARNT) have been coexpressed in the yeast, *Saccharomyces cerevisiae*, with specific transcription activation assessed by β-galactosidase activity produced from a reporter plasmid [9]. Coexpression of human AHR and ARNT lead to the transcription of a DRE-containing reporter plasmid in the absence of exogenous AHR agonists. Critically, the majority of PCB congeners are likely to interact with AHR in their non-planar conformations, meaning that they are dioxin-like and are likely to be recognized as dioxins by the AHR complex. When activated by a dioxin, or, in the case of the study, a dioxin-like PCB, the AHR translocates into the nucleus and dimerizes with the ARNT protein to form the AHR/ARNT complex, which then binds to a specific DNA recognition site within a nucleus: the DRE.

**Response**

To respond to the signal:

* AhR/ARNT complex in the nucleus binds to 7 DREs (DNA recognition site) in the CYP1A1 promoter
* CYP1A1 promoter activates CYP1A1 gene fused to the Akaluc luciferase reporter

When activated by dioxin, the cytosolic AhR protein complex translocates into the nucleus and dimerizes with the ARNT (Ah receptor nuclear translocator) protein. The heteromeric ligand:AhR/Arnt complex then recognizes and binds to its specific DNA recognition site, the dioxin response element (DRE). DREs are located upstream of cytochrome P4501A1 (CYP1A1) and other AhR-responsive genes, and binding of the AhR complex stimulates their transcription. Although CYP1A1 expression has been used as the model system to define the biochemical and molecular mechanism of AhR action, there is still limited knowledge about the roles of each of the seven DREs located in the CYP1A1 promoter.

![Construct diagram showing a CYP1A1 Promoter containing DREs upstream of the Akaluc luciferase reporter](https://static.igem.wiki/teams/4458/wiki/response.png)

Construct design for our PCB Responder

#### Build

To construct our device, we used Gibson Assembly to create our final plasmid pLK001 that contained:

* Digested pBF3038
* CYP1A1 gene fragments
* Human CYP1A1 promoter region
* Akaluc

#### Test

We assembled our parts using Gibson Assembly and were able to successfully get colonies!

![A picture of successfully transformed chassis with the Akaluc plasmid insertion](https://static.igem.wiki/teams/4458/wiki/gibson-plate.png)

Successfully transformed chassis with the Akaluc plasmid insertion

#### Iteration

[1] Bedard, D. L. (2014). PCB dechlorinases revealed at last. Proceedings of the National Academy of Sciences, 111(33), 11919–11920. https://doi.org/10.1073/pnas.1412286111

[2] Wang, S., Chng, K. R., Wilm, A., Zhao, S., Yang, K.-L., Nagarajan, N., & He, J. (2014). Genomic characterization of three unique dehalococcoides that respire on persistent polychlorinated biphenyls. Proceedings of the National Academy of Sciences, 111(33), 12103–12108. https://doi.org/10.1073/pnas.1404845111

[3] GenSmart™ Codon Optimization Tool-GenScript. (n.d.). Retrieved October 14, 2022, from https://www.genscript.com/gensmart-free-gene-codon-optimization.html

[4] Round, J. W., Robeck, L. D., & Eltis, L. D. (2021). An integrative toolbox for synthetic biology in Rhodococcus. ACS Synthetic Biology, 10(9), 2383–2395. https://doi.org/10.1021/acssynbio.1c00292

[5] Chobtang, J., De Boer, I. J., Hoogenboom, R. L., Haasnoot, W., Kijlstra, A., & Meerburg, B. G. (2011). The need and potential of biosensors to detect dioxins and dioxin-like polychlorinated biphenyls along the milk, eggs and meat food chain. Sensors, 11(12), 11692–11716. https://doi.org/10.3390/s111211692

[6] Hornbuckle, K., & Robertson, L. (2010). Polychlorinated biphenyls (PCBS): Sources, exposures, toxicities. Environmental Science &Technology, 44(8), 2749–2751. https://doi.org/10.1021/es100801f

[7] Liu, X., Germaine, K., Ryan, D., & Dowling, D. (2010). Whole-cell fluorescent biosensors for bioavailability and biodegradation of polychlorinated biphenyls. Sensors, 10(2), 1377–1398. https://doi.org/10.3390/s100201377

[8] Miller, C. A. (1997). Expression of the human aryl hydrocarbon receptor complex in yeast. Journal of Biological Chemistry, 272(52), 32824–32829. https://doi.org/10.1074/jbc.272.52.32824

[9] Environmental Protection Agency. (2019, October 4). Guidance for Applicants Requesting to Treat/Dispose of PCBs Using Incineration or an Alternative Method. Regulations.gov. Retrieved October 13, 2022, from https://www.regulations.gov/document/EPA-HQ-OLEM-2018-0305-0010

## Experiments

In order to test whether or not our solution would work, we had to conduct some experiments on the effectiveness of the bacteria. We conducted these experiments at Genspace and BUGSS, with each location focusing on different parts of the project.

### Genspace

The experiments at Genspace focused on creating a bacteria which would bioluminesce in the presence of PCBs.

#### Experiments

The goal of this part of the project was to transfer a plasmid containing the Akaluc construct into the chassis of choice, which was yeast. Towards the end of the project, we also focused on increasing the visibility of the bioluminescence, though we did not get very far on that.

#### Results

Through repeated ligations, purifications, transformations, and platings, we eventually achieved cells which contained the desired gene.

### BUGSS

The experiments at BUGSS focused on the other aspect of the project, that being the degradation of PCBs into less harmful products.

#### Experiments

This portion was cloned a plasmid in *E. coli* and then transferred it into *Rhodococcus jostii* as a chassis, since it can survive in water and on land.

#### Results

We were able to successfully clone pcbA4 into *E. coli* as shown by the band of ~1500 bp on a colony screening PCR gel.

![A agarose gel showing the results of colony PCR on our putative pcbA4 colonies. DNA ladder bands on the left indicate that the colony band on the right is about 1500bp in length.](https://static.igem.wiki/teams/4458/wiki/picture4.png)

DNA agarose gel of PCR screening on our putative pcbA4 colonies

Due to time constraints we did not test the efficacy of our degradation genes, pcbA1, pcbA4, and pcbA5. If we were to continue this project in the future, we would experiment with the reaction rates of those enzymes on the breakdown of PCBs.

## Experiments on *Rhodococcus*

Because *Rhodococcus spp.* do not have a lot of documentation in iGEM, we also focused on characterizing the bacteria. In addition to transforming the bacteria to include our desired genes, we also tested for its optimal growth conditions and growth rate. We ran each test multiple times, changing conditions slightly each time, in order to identify the best environment for this bacteria.

#### Results with *Rhodococcus*

From our experiments with *Rhodococcus jostii*, we have learned a lot more about this species of bacteria. First, it seems to prefer a temperature of 30 degrees Celsius, which is slightly colder than *E. coli*. Finally, it has a doubling time of about 190 minutes. Also see our [Contribution page](https://2022.igem.wiki/east-coast-biocrew/contribution) for more characterization of *Rhodococcus* growth.

![Graph of Rhodococcus growth at 30 degrees celsius over 5 hours” style=](https://static.igem.wiki/teams/4458/wiki/picture1.png)

Graph of Rhodococcus growth at 30 degrees celsius over 5 hours

{% 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 %}

## What should this page contain?

---

* Clearly and objectively describe the results of your work.
* Future plans for the project.
* Considerations for replicating the experiments.

## Describe what your results mean

---

* Interpretation of the results obtained during your project. Don't just show a plot/figure/graph/other, tell us what you think the data means. This is an important part of your project that the judges will look for.
* Show data, but remember **all measurement and characterization data must also be on the Part's Main Page on the [Registry](http://parts.igem.org/Main_Page).** Otherwise these data will not be in consideration for any medals or part awards!
* Consider including an analysis summary section to discuss what your results mean. Judges like to read what you think your data means, beyond all the data you have acquired during your project.

## Project Achievements

---

You can also include a list of bullet points (and links) of the successes and failures you have had over your summer. It is a quick reference page for the judges to see what you achieved during your summer.

* A list of linked bullet points of the successful results during your project
* A list of linked bullet points of the unsuccessful results during your project. This is about being scientifically honest. If you worked on an area for a long time with no success, tell us so we know where you put your effort.

## Inspirations

---

* [2019 Newcastle](https://2019.igem.org/Team%3ANewcastle/Results)
* [2019 Munich](https://2019.igem.org/Team%3AMunich/Results)
* [2019 Tec Chihuahua](https://2019.igem.org/Team%3ATec-Chihuahua/Results)
* [2020 Aalto Helsinki](https://2020.igem.org/Team%3AAalto-Helsinki/Results)
* [2020 GreatBay SCIE](https://2020.igem.org/Team%3AGreatBay_SCIE/Results)
* [2020 Queens Canada](https://2020.igem.org/Team%3AQueens_Canada/Results)

{% endblock %}