iGEM 2018 — Team UGA

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

# Team:UGA/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%3AUGA)
[HOME](https://2018.igem.org/Team%3AUGA)

TEAM

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

PROJECT

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

PARTS

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

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

HUMAN PRACTICES

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

AWARDS

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

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

# Notebook

##### May 7th-11th

gBlocks containing Gal1-BS3 and Gal1-AmilC were obtained and resuspended in water.
gBlocks containing Scfv1 and Scfv2 (not used in final project design) were received and resuspended.

##### May 14th-18th

LB and gentamycin plates were made. LB broth was made.
Competent Dh5a cells were created and tested using pdonor 207 stock plasmid.

##### May 21st-25th

The pdonor 207 plasmid was miniprepped.
PGWB1 and PGWB2 vectors were miniprepped.
Gateway cloning of all constructs into pdonor 207 was attempted using the combined BP/LR reaction. No colonies were observed.

##### May 28th-June 1st

Gblocks were all gateway cloned (BP) into the pdonor 207 plasmids. Colonies were yielded on each plate. Primers designed for validation of the parts in pdonor 207.

##### June 4th-June 8th

The pdonor plasmids were sent off for sequencing and sequences were confirmed.
Gateway cloning LR reactions were performed using the PGWB1 and PGWB2 plasmids, along with AmilC, BS3, Scfv1, and Scfv2.
Colonies observed.

##### June 11th-June 15th

Colonies mini-prepped using the GENEjet mini prep kit.
PGWB1 and PGWB2 plasmids were screened using Vspi, incorrect except for Gal1 BS3 PGWB1.
Began planning first experiments of agrobacterium into Nicotiana benthamiana—because our system would be using toxin, we would first need a proof of concept showing we can transfer genes into plants.
Sponsoring PI reached out to Jennifer Brophy, who provided minimal CaMV 35S promoter with 6X UAS and VP-16 Gal4 activator plasmids in agrobacterium-compatible plasmids.

##### June 18th-June 22nd

Attempted to design plasmid encoding 6X UAS CaMV 35S promoter, but complexities were caused by repeated sequences that prevented gBlock synthesis
BS3 and AmilC cloning attempts into PGWB2 were conducted, no colonies again.
Gateway compatible multiple cloning site (7X RE) was designed and ordered as a gBlock.

##### June 25th-June 29:

Gateway compatible 7X RE was cloned into pDonor 207 plasmid using BP clonase.
Clones were validated using Kpni and Bglii performed, 7X RE was successfully cloned.
Primers designed to add 5’ site of Kpni to the CaMV 35S 6XUAS promoter and Asci to the 3’ end, as well as Xbai and Spei sites.. gBlocks for BS3 and AmilC created with Asci at the 5’ end and Spei at the 3’ end.

##### July 2nd-July 6th

The 6X UAS sequences were PCR amplified using Xbai and Spei forward and reverse primers, as well as kpni and asci 5’ addition and 3’ addition primers, and purified through a Genejet column.
The amplified fragments were digested using the handles for each primer that were added. The 7X RE plasmid was also digested with the same sets of restriction enzymes.

##### July 9th-13th

The fragments containing the amplified 6X UAS promoter sequences were gel purified and the DNA was extracted using the Genejet purification kit.
The promoters were ligated and transformed into 7X RE plasmid. A negative control was included.
Colonies observed on all plates.

##### July 16th-July 20th

The sequences plasmids were digested with Vspi to screen. They all returned as backbone.
We performed the interlab study. We helped Lambert to perform the interlab following our experience with it.

##### July 23rd-August 3rd

Cloning steps from July 2nd to July 13th were performed again, and yielded the 6X UAS promoter 7XRE plasmid.

##### August 6th-10th

In the interests of time, we decided to begin attempting to clone using the 6X UAS – CaMV 35S –GFP construct, pJAB 1504, and the VP-16 plasmid, pJAB 1489. BS3 PGWB1 was also transformed. First Agrobacterium cloning method only yielded pJAB 1489 colonies on the Rifampicin-Kan plates. Briefly, the transformations were incubated with DNA for 10 minutes, followed by flash freezing in liquid nitrogen. A 4 minute recovery in 1 mL of LB at 37 degrees was carried out followed by 4 hours of shaking at 28 degrees. Cultures were plated on Rifampicin and Kanamycin plates for 2-3 days before checking for colonies.

##### August 13th-17th

Agrobacterium transformations from August 6th-10th were repeated, but again did not yield colonies. A longer, 5 minute flash freeze was carried out, and BS3 yielded colonies.

##### August 20th-24th

From August 13th to 17th, we got down to our last stock of EHA105, so for this week we prepared competent cells for EHA105.
Interlab CFU was repeated.

##### August 27th-31st

Again, The experiment from the 13th-17th was performed, and finally the pJAB1504 plasmid was cloned.

##### September 3rd-14th

For the 6XUAS promoter and BS3 resistance gene plasmids, they were cloned into the shipping backbone using Xbai and Spei sites.

##### September 17th-28th

Transient Expression of pJAB1504, pJAB 1489, and BS3 experiments were performed in Nicotiana benthamiana. Fluorescence and leaf death pictures were taken.

##### October 1st-5th

Helped Lamber test electroporator as part of collaborations.

##### October 8th-12th

Plate reader measurements taken for Lambert collaboration and Wiki design. Parts submitted.


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

# Team:UGA/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%3AUGA)
[HOME](https://2018.igem.org/Team%3AUGA)

TEAM

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

PROJECT

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

PARTS

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

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

HUMAN PRACTICES

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

AWARDS

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

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

# Experiments

The following procedures were used to transform four conditions of plasmid into Nicotiana benthamiana.
Plasmids are as follows:
pJAB1489 (VP-16 driven by CaMV 35S promoter)
PJAB 1504 (6X UAS enhancing a minimal CaMV 35S promoter)

Gal1-BS3 (Gal1 promoter driving BS3 in PGWB1 backbone)

Condition 1: pJAB 1504+pJAB1489
Condition 2: Gal1-BS3
Condition 3: Agrobacterium EHA105 only
Condition 4: Induction buffer only

# Transformation of EHA105 Agrobacterium

1. Inoculate 2.0 mL of LB with desired colony of Agrobacterium, incubate at 28°C overnight
2. Cool on ice for 10 min
3. Pellet at 4°C at 1500xg for 5 min
4. Resuspend in 100μL of cold, sterile 20mM CaCl2
5. Add 2.0 μL of purified plasmid (about 1μg of DNA)
6. Freeze in liquid nitrogen
7. Incubate at 37°C for 4 min
8. Dilute in 1 mL of LB media and recover at 28°C shaking at 200 rpm for 4 hours
9. Pellet and spread on LB with selective media (rifampicin and kanamycin). Incubate at 28°C for 36-48 hrs

# Agrobacterium Transient Expression

1. Grow 1mL overnight culture of Agrobacterium containing desired expression vector in LB+ appropriate antibiotic
2. Pellet 0.2mL of overnight culture at 4000xg for 5 min
3. Decant media and resuspend pellet in 0.5mL of induction buffer or sterile water
4. Measure cell density at OD600
5. Dilute cell density to OD600 = 0.05 with induction buffer or sterile water
6. Hand infiltrate N. benthamiana leaves with 0.5mL of bacterial suspension using a 1mL blunt syringe
7. Check GFP expression at 2 and 4 days, and BS3 expression at 4 days.

# Induction Buffer Recipe

39.45mL sterile water
5mL sterile 500mM MES
5mL sterile 5% glucose
0.5mL sterile NaH2PO4
50μL sterile 200mM Acetosyringone

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

# Team:UGA/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%3AUGA)
[HOME](https://2018.igem.org/Team%3AUGA)

TEAM

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

PROJECT

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

PARTS

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

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

HUMAN PRACTICES

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

AWARDS

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

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

# Results

|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| ![](<table style=)| VP-16 (pjab1489) and 6XUAS-CaMV35S-GFP (pjab 1504) | | ![Stephan]( https://static.igem.org/mediawiki/2018/8/83/T--UGA--white_1.jpg) | ![Stephan]( https://static.igem.org/mediawiki/2018/6/64/T--UGA--14891504d1g.jpg ) | ![Stephan]( https://static.igem.org/mediawiki/2018/3/39/T--UGA--4dw214891504.jpg) | ![Stephan]( https://static.igem.org/mediawiki/2018/7/7e/T--UGA--4dg2148915042.jpeg) | | VP-16 and 6X UAS-CaMV minimal 35S-GFP, white, photographed after 2 days | VP-16 and 6X UAS-CaMV minimal 35S-GFP, green, photographed after 2 days | VP-16 and 6X UAS-CaMV minimal 35S-GFP, white, photographed after 4 days | VP-16 and 6X UAS-CaMV minimal 35S-GFP, green, photographed after 4 days | | Agrobacterium Only | | ![](<table style=)| ![Stephan]( https://static.igem.org/mediawiki/2018/2/2d/T--UGA--agro2dw.jpg) | ![Stephan]( https://static.igem.org/mediawiki/2018/6/64/T--UGA--14891504d1g.jpg ) | ![Stephan]( https://static.igem.org/mediawiki/2018/3/38/T--UGA--d4w_agro.jpg) | ![Stephan]( https://static.igem.org/mediawiki/2018/d/d6/T--UGA--agro4dg.jpg) | | Agrobacterium only, white, photographed after 2 days | Agrobacterium only, green, photographed after 2 days | Agrobacterium only, white, photographed after 4 days | Agrobacterium only, green, photographed after 4 days |  ![](<table style=)| Induction Buffer | || ![Stephan]( https://static.igem.org/mediawiki/2018/3/34/T--UGA--IBw1_2d.jpg ) | ![Stephan]( https://static.igem.org/mediawiki/2018/8/82/T--UGA--green_2_day_id.jpeg) | ![Stephan]( https://static.igem.org/mediawiki/2018/d/da/T--UGA--4dib.jpeg) | ![Stephan]( https://static.igem.org/mediawiki/2018/7/75/T--UGA--g4dib.jpg) | | Induction Buffer, white, photographed after 2 days | Induction Buffer, green, photographed after 2 days | Induction Buffer, white, photographed after 4 days | Induction Buffer, green, photographed after 4 days | | pGal1 BS3 and VP-16|  |  | | --- | --- | | ![Stephan]( https://static.igem.org/mediawiki/2018/a/ac/T--UGA--BS3.png) | ![Stephan]( https://static.igem.org/mediawiki/2018/8/8f/T--UGA--Agro_only.png) | | pGal1—BS3 and pJAB1489 after 4 days | Agrobacterium only after 4 days |  Brief Summary of Results  Following transient expression of pJAB1489 and pJAB1504, there appeared to be little to no expression of GFP compared to the background level of expression. There also appeared to be no expression of the BS3 construct either. As neither construct showed expression, future experiments should be focused on validating the system in Nicotiana benthamiana, optimizing the gene transfer method.    Future Directions  Following validation, the system may be adapted to create an inducible toxin recognition system. As described by Tsinghua 2017, the single chain variable fragments can be attached to the activating and binding domains of Gal4 to enhance expression of genes downstream of a UAS/promoter. It is our hope that future iGEM teams will be able to harness this type of system to create a variety of toxin recognition systems to remove contaminated food before it hits the shelves.  The 6X UAS promoter is part BBa\_K2575001   The BS3 part number is: BBa\_K2575000 | | |