iGEM 2018 — Team ULaVerne_Collab

[![](https://static.igem.org/mediawiki/2018/8/8b/HQ_page_logo.jpg)](https://2018.igem.org)

# Team:ULaVerne Collab/Notebook

![](https://static.igem.org/mediawiki/2018/4/4f/2018_igem_mobile_menu_logo.svg)

![](https://static.igem.org/mediawiki/igem.org/3/3b/Menu_icon.svg)

[![](http://placehold.it/350x150/e4dede/c4baba)](https://2018.igem.org/Team%3AULaVerne_Collab)
[HOME](https://2018.igem.org/Team%3AULaVerne_Collab)

TEAM

[Team Members](https://2018.igem.org/Team%3AULaVerne_Collab/Team)
[Collaborations](https://2018.igem.org/Team%3AULaVerne_Collab/Collaborations)

PROJECT

[Description](https://2018.igem.org/Team%3AULaVerne_Collab/Description)
[Design](https://2018.igem.org/Team%3AULaVerne_Collab/Design)
[Experiments](https://2018.igem.org/Team%3AULaVerne_Collab/Experiments)
[Notebook](https://2018.igem.org/Team%3AULaVerne_Collab/Notebook)
[InterLab](https://2018.igem.org/Team%3AULaVerne_Collab/InterLab)
[Model](https://2018.igem.org/Team%3AULaVerne_Collab/Model)
[Results](https://2018.igem.org/Team%3AULaVerne_Collab/Results)
[Demonstrate](https://2018.igem.org/Team%3AULaVerne_Collab/Demonstrate)
[Improve](https://2018.igem.org/Team%3AULaVerne_Collab/Improve)
[Attributions](https://2018.igem.org/Team%3AULaVerne_Collab/Attributions)

PARTS

[Parts Overview](https://2018.igem.org/Team%3AULaVerne_Collab/Parts)
[Basic Parts](https://2018.igem.org/Team%3AULaVerne_Collab/Basic_Part)
[Composite Parts](https://2018.igem.org/Team%3AULaVerne_Collab/Composite_Part)
[Part Collection](https://2018.igem.org/Team%3AULaVerne_Collab/Part_Collection)

[SAFETY](https://2018.igem.org/Team%3AULaVerne_Collab/Safety)

HUMAN PRACTICES

[Human Practices](https://2018.igem.org/Team%3AULaVerne_Collab/Human_Practices)
[Education & Engagement](https://2018.igem.org/Team%3AULaVerne_Collab/Public_Engagement)

AWARDS

[Applied Design](https://2018.igem.org/Team%3AULaVerne_Collab/Applied_Design)
[Entrepreneurship](https://2018.igem.org/Team%3AULaVerne_Collab/Entrepreneurship)
[Hardware](https://2018.igem.org/Team%3AULaVerne_Collab/Hardware)
[Measurement](https://2018.igem.org/Team%3AULaVerne_Collab/Measurement)
[Model](https://2018.igem.org/Team%3AULaVerne_Collab/Model)
[Plant](https://2018.igem.org/Team%3AULaVerne_Collab/Plant)
[Software](https://2018.igem.org/Team%3AULaVerne_Collab/Software)

[JUDGING FORM ⇗](https://igem.org/2018_Judging_Form?team=ULaVerne_Collab)

# Notebook

# June

Week 2

(6/12) Restriction Digestion of PETase and K1357009 - Vector

(6/13) Restriction Digestion Old Vector K1357009 + pSB1C3
Ligation of PCR Purified Products
Transformation of Ligation rxns

(6/14) Transformation
Commercial Cells:
1. 1uL of Ligated Vector and Insert
Non-Comm:
2. 1 uL Ligated Vector and Insert
3. 5 uL Ligated Vector and Insert
4. 1 uL Vector only
5. 5 uL Vector only

Week 3

(6/18) Overnight Colonies of Transformations
Plan to pick 4+ colonies to screen ligations
(more depending on how many colonies were on your control ligation plate.) Pick 2+ colonies when
growing up bacteria for DNA stocks.

(6/19) Miniprep, Restriction Digest of Miniprep & GEL, Digestion of Promoter J23110

(6/21) Digestion Constitutive Promoter J23110, PCR Amplification of PETase

(6/22) GEL Purification, Restriction Digestion of PeTase and K1357009

Week 4

(6/25) Ligation, Transformation
Results
PeTase + promoter control 1 uL - 19
PeTase + promoter ligation 1uL - 30
PeTase + promoter control 5 uL - 40
PeTase + promoter ligation 5 uL - 54
Colonies
PeTase +promoter 1 uL- 8 colonies
PeTase + promoter 5 uL - 10 colonies

(6/26) Overnights Colonies of Transformation

(6/27) Miniprep

(6/29) Electroporation, Nanodrop

# July

Week 1

(7/2) Gel Electrophoresis of Digested Vector w/ E + X, ElectroTransformation Data

Week 2

(7/12) Electrocompetent Cells 2, Digestion of PETase/ K1357009 from (5/29): Miniprep #2, #5, #6, NanoDrops of 5/29 Minipreps: PETase & K1357009, Gel Electrophoresis of PETase/ K1357009 from (5/29) and J23110 from Registry

![](https://static.igem.org/mediawiki/2018/d/d8/T--ULaVerne_Collab--IMG_6923.jpeg)

Week 3

(7/16) Digestion of PETase/ K1357009 from (5/29): Miniprep #1 #4, #8

(7/17) Electro Competent Cells, Nanodrop of Gel Purification

![](https://static.igem.org/mediawiki/2018/6/64/T--ULaVerne_Collab--Electro_comp.jpeg)

(7/18) Ligation Calculation & Procedure, PCR Purification of Ligation Mixtures, ElectroTransformation, Mini Prep

(07/19) Chemical transformation of Ligations, Nanodrop PETase/ K1357009 \*from (5/29)\*: Miniprep #1 #4, Digestion (high DNA), Gel Electrophoresis, Gel Purification

(7/20) Ligation Reaction of J23110 Vector + PETase/K1357009

Week 4

(7/23) Interlab study, picking colonies from plates

(7/24) Miniprep, Nanodrops

(7/25) Miniprep, Nanodrop of Mini Prep PETase Circuit (Attempt 1), Digest of PETase Circuit (Attempt 1)

(7/26) Miniprep the Overnight, Nanodrop of MP, Digestions J23 and PET/K13

Week 5

(7/30) PCR Reaction, Primer Rehydration, Dilution of Promoter Stock, Promoter PCR, mHETase PCR, Modified Petase PCR for Cutinase test circuit, Modified Petase PCR for mHETase test circuit, Nanodrop

(7/31) Gibson Assembly Reaction 1 and PCR Reaction 2, Q5 2X PCR Reaction, Amil-CP Reaction (2), Curli Non Gibson PCR, OLD PETase PCR, Promoter Reactions

# August

Week 1

(8/1) PCR products so far,

(8/2) PCR Purification of Curli Non-Gibson, Nanodrop Curli Non-Gibson, curli PCR for Gibson, Promoter PCR , Old PETase gel, PCR products

(8/3) Curli PCR Gibson redo sorry, Promoter Attempt 3,

Week 2

(8/7) Gibson Assembly, Cutinase Test circuit, PETase control circuit, mHETase test circuit, PCR Reaction, Cutinase for Old PETase,

(8/8) Gibson assembly of PETase mHet circuit,

(8/9) Mini Prep, Nanodrop of Gibson Mini Preps, Digestions Gibson Mini Preps (20 uL rxn)

(8/10) Mini Prep, Nanodrop oF Gibson Mini Preps, Digestions, Digestion #2

Week 3

(8/13) Digestion Gibson Mini Preps (20 uL rxn ), Gel, Pick colonies

(8/14) Nanodrop, miniprep of gibson parts, Digestions, Gel

(8/15) Nanodrop

Week 4

(8/20) PCR Tests, CUT mHet test CUT pcr, Old Pet CTRL PCR,

(8/21) Miniprep CUT test, nanodrop CUT test, PCR CUT Test CUT 1, CUT test CUT 2

# September

Week 1

(9/4) Beginning of characterization

(9/7) Sodium Dodecyl Sulphate PolyAcrylamide Gel Electrophoresis

(09/08) Dilutions for Protein Gel

Week 2

(9/12) Sample Prep for Protein Gel

(9/15) International Coastal Cleanup

Week 3

(9/20) Sample Prep 2 for Protein Gel

(9/21) Staining and Destaining

Week 4

(9/25) Protein Extraction

(9/27) Math Modeling

# October

Week 1

(10/1) Begin working on Wiki


[![](https://static.igem.org/mediawiki/2018/8/8b/HQ_page_logo.jpg)](https://2018.igem.org)

# Team:ULaVerne Collab/Experiments

![](https://static.igem.org/mediawiki/2018/4/4f/2018_igem_mobile_menu_logo.svg)

![](https://static.igem.org/mediawiki/igem.org/3/3b/Menu_icon.svg)

[![](http://placehold.it/350x150/e4dede/c4baba)](https://2018.igem.org/Team%3AULaVerne_Collab)
[HOME](https://2018.igem.org/Team%3AULaVerne_Collab)

TEAM

[Team Members](https://2018.igem.org/Team%3AULaVerne_Collab/Team)
[Collaborations](https://2018.igem.org/Team%3AULaVerne_Collab/Collaborations)

PROJECT

[Description](https://2018.igem.org/Team%3AULaVerne_Collab/Description)
[Design](https://2018.igem.org/Team%3AULaVerne_Collab/Design)
[Experiments](https://2018.igem.org/Team%3AULaVerne_Collab/Experiments)
[Notebook](https://2018.igem.org/Team%3AULaVerne_Collab/Notebook)
[InterLab](https://2018.igem.org/Team%3AULaVerne_Collab/InterLab)
[Model](https://2018.igem.org/Team%3AULaVerne_Collab/Model)
[Results](https://2018.igem.org/Team%3AULaVerne_Collab/Results)
[Demonstrate](https://2018.igem.org/Team%3AULaVerne_Collab/Demonstrate)
[Improve](https://2018.igem.org/Team%3AULaVerne_Collab/Improve)
[Attributions](https://2018.igem.org/Team%3AULaVerne_Collab/Attributions)

PARTS

[Parts Overview](https://2018.igem.org/Team%3AULaVerne_Collab/Parts)
[Basic Parts](https://2018.igem.org/Team%3AULaVerne_Collab/Basic_Part)
[Composite Parts](https://2018.igem.org/Team%3AULaVerne_Collab/Composite_Part)
[Part Collection](https://2018.igem.org/Team%3AULaVerne_Collab/Part_Collection)

[SAFETY](https://2018.igem.org/Team%3AULaVerne_Collab/Safety)

HUMAN PRACTICES

[Human Practices](https://2018.igem.org/Team%3AULaVerne_Collab/Human_Practices)
[Education & Engagement](https://2018.igem.org/Team%3AULaVerne_Collab/Public_Engagement)

AWARDS

[Applied Design](https://2018.igem.org/Team%3AULaVerne_Collab/Applied_Design)
[Entrepreneurship](https://2018.igem.org/Team%3AULaVerne_Collab/Entrepreneurship)
[Hardware](https://2018.igem.org/Team%3AULaVerne_Collab/Hardware)
[Measurement](https://2018.igem.org/Team%3AULaVerne_Collab/Measurement)
[Model](https://2018.igem.org/Team%3AULaVerne_Collab/Model)
[Plant](https://2018.igem.org/Team%3AULaVerne_Collab/Plant)
[Software](https://2018.igem.org/Team%3AULaVerne_Collab/Software)

[JUDGING FORM ⇗](https://igem.org/2018_Judging_Form?team=ULaVerne_Collab)

# Experiments

# Gibson Assembly

1.Set up the following reaction on ice:

|  | 2-3 Fragment Assembly | 4-6 Fragment Assembly | Positive Control\*\* |
| --- | --- | --- | --- |
| Total Amount of Fragments | 0.02–0.5 pmols\* X μl | 0.2–1 pmols\* X μl | 10 μl |
| Gibson Assembly Master Mix (2X) | 10 μl | 10 μl | 10 μl |
| Deionized H2O | 10-X μl | 10-X μl | 0 |
| Total Volume | 20 μl\*\*\* | 20 μl\*\*\* | 20 μl |

\* Optimized cloning efficiency is 50–100 ng of vectors with 2–3 fold of excess inserts. Use 5 times more of inserts if size is less than 200 bps. Total volume of unpurified PCR fragments in Gibson Assembly reaction should not exceed 20%.
\*\* Control reagents are provided for 5 experiments.
\*\*\* If greater numbers of fragments are assembled, additional Gibson Assembly Master Mix may
be required.

2.Incubate samples in a thermocycler at 50°C for 15 minutes when 2 or 3 fragments are being assembled or 60 minutes when 4-6 fragments are being assembled. Following incubation, store samples on ice or at -20°C for subsequent transformation.

# Gel Electrophoresis

500 mL of TAE Buffer

1. Add 10 mL of TAE buffer

2. 490 mL of D.I. water

1% gel

1. Add 0.5 g of Agarose

2. Add 50 mL of TAE

3. Microwave for 1 min and 45 sec

4. Let it sit until room temperature

5. Add 5 uL of SYBR Safe DNA Gel Stain

6. Swirl Solution

7. Pour gel into cast

8. Let it sit for 20 minutes.

Loading of gel

1. Well 1: 6uL of the 1Kb ladder or 5uL of the APEX ladder

2. Other wells: 6 uL of your DNA (5 parts DNA, 1 part Dye)

# SDS PAGE: Protein

8% Gel - Resolving

Add 4ml of 30% Acrylamide/bis 0.5M

Add7.03 ml of Deionized water

Add 3.75 ml( pH 8.8)

Add 150 uL 10% SDS

Add 75 uL 10% APS

Add 7.5 uL of TEMED

Approximately 15ml

5% Stacking

Add 1.3 ml of 30% Acrylamide/bis 0.5M

Add 5.5 ml of Deionized water

Add 1ml of Tris (use pH 6.8)

Add 80 ul of 10% SDS

Add 80ul of 10% APS

Add 8ul of TEMED

Plastic Weighing: We weighed the plastic particles with the laboratory weighing scale before and after inoculation with transformed E. coli+Media.

# Scanning Electron Microscopy: This is part of our future directions.

[![](https://static.igem.org/mediawiki/2018/8/8b/HQ_page_logo.jpg)](https://2018.igem.org)

# Team:ULaVerne Collab/Results

![](https://static.igem.org/mediawiki/2018/4/4f/2018_igem_mobile_menu_logo.svg)

![](https://static.igem.org/mediawiki/igem.org/3/3b/Menu_icon.svg)

[![](http://placehold.it/350x150/e4dede/c4baba)](https://2018.igem.org/Team%3AULaVerne_Collab)
[HOME](https://2018.igem.org/Team%3AULaVerne_Collab)

TEAM

[Team Members](https://2018.igem.org/Team%3AULaVerne_Collab/Team)
[Collaborations](https://2018.igem.org/Team%3AULaVerne_Collab/Collaborations)

PROJECT

[Description](https://2018.igem.org/Team%3AULaVerne_Collab/Description)
[Design](https://2018.igem.org/Team%3AULaVerne_Collab/Design)
[Experiments](https://2018.igem.org/Team%3AULaVerne_Collab/Experiments)
[Notebook](https://2018.igem.org/Team%3AULaVerne_Collab/Notebook)
[InterLab](https://2018.igem.org/Team%3AULaVerne_Collab/InterLab)
[Model](https://2018.igem.org/Team%3AULaVerne_Collab/Model)
[Results](https://2018.igem.org/Team%3AULaVerne_Collab/Results)
[Demonstrate](https://2018.igem.org/Team%3AULaVerne_Collab/Demonstrate)
[Improve](https://2018.igem.org/Team%3AULaVerne_Collab/Improve)
[Attributions](https://2018.igem.org/Team%3AULaVerne_Collab/Attributions)

PARTS

[Parts Overview](https://2018.igem.org/Team%3AULaVerne_Collab/Parts)
[Basic Parts](https://2018.igem.org/Team%3AULaVerne_Collab/Basic_Part)
[Composite Parts](https://2018.igem.org/Team%3AULaVerne_Collab/Composite_Part)
[Part Collection](https://2018.igem.org/Team%3AULaVerne_Collab/Part_Collection)

[SAFETY](https://2018.igem.org/Team%3AULaVerne_Collab/Safety)

HUMAN PRACTICES

[Human Practices](https://2018.igem.org/Team%3AULaVerne_Collab/Human_Practices)
[Education & Engagement](https://2018.igem.org/Team%3AULaVerne_Collab/Public_Engagement)

AWARDS

[Applied Design](https://2018.igem.org/Team%3AULaVerne_Collab/Applied_Design)
[Entrepreneurship](https://2018.igem.org/Team%3AULaVerne_Collab/Entrepreneurship)
[Hardware](https://2018.igem.org/Team%3AULaVerne_Collab/Hardware)
[Measurement](https://2018.igem.org/Team%3AULaVerne_Collab/Measurement)
[Model](https://2018.igem.org/Team%3AULaVerne_Collab/Model)
[Plant](https://2018.igem.org/Team%3AULaVerne_Collab/Plant)
[Software](https://2018.igem.org/Team%3AULaVerne_Collab/Software)

[JUDGING FORM ⇗](https://igem.org/2018_Judging_Form?team=ULaVerne_Collab)

# Results

### Current Stage of Results:

* We were able to successfully add our parts to our e.coli
* Still in the process of testing and looking for actual degradation.
* Aim to take SEM images to look for nano sized missing pockets on the plastics surface.

### DNA assembly!

![](https://static.igem.org/mediawiki/2018/2/25/T--ULaVerne_Collab--fig1.png)* Figure 1: PCR products creating overlapping ends for the Gibson assembly of our parts. K1357009 is our amilCP and double terminator part at 874 bp. The two PETase enzymes are around 1000 bp each with slight variation because of the difference in overlapping ends between parts. mHETase is around 2000 bp and the promoter and vector is also near 2000.
![](https://static.igem.org/mediawiki/2018/0/0b/T--ULaVerne_Collab--fig_2.png)* Figure 2: Plates showing blue colonies on plates for each of our circuits, proving that amilCP reporter is active and that the DNA has been assembled properly.
![](https://static.igem.org/mediawiki/2018/b/b5/T--UlaVerne_Collab--DNAAssembly3.jpg)* Figure 3: Assembled parts were digested to ensure proper DNA lengths in the circuits. The first gel shows the mHETase circuit digested once in lane one which made a band at 6000 bp, and once digested with two enzymes showed bands at 2000 bp for the vector and 4000 bp for the assembled circuit of mHETase, mutPETase, and the k1357009 reporter. The second and third gels show the PETase circuits digested with one enzyme at about 4000 bp, and then when digested with two it showed two bands very near to 2000 bp which is where the vector and the assembled circuit of the PETase and k1357009 will be.
![](https://static.igem.org/mediawiki/2018/5/54/T--ULaVerne_Collab--DNAAssembly4.jpg)* Figure 4: After assembly and transformation, PCR reactions were done on the mini prepped plasmids to ensure that the circuits contained the proper enzymes. In order from left to right, the mutPETase from mHET circuit, control PETase from control circuit, mHETase from mHETase circuit, and mutPETase from mutPETase circuit.

These results conclude that we cannot utilize this method to degrade plastic at real time with the current rate of the mutant PETase. We aim to further characterize with SEM to understand if the Mutant PETase is better than the Wild Type.

# Data Weight of Plastic over Time

![](https://static.igem.org/mediawiki/2018/f/fb/T--ULaVerne_Collab--DataTableWeight.jpg)