iGEM 2021 — Team UZurich

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# Team:UZurich/Notebook

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# Notebook

href

#### July

| Mo | Tu | We | Th | Fr | Sa | Su |
| --- | --- | --- | --- | --- | --- | --- |
| [28](#0628) | 29 | [30](#0630) | [1](#0701) | [2](#0702) | 3 | 4 |
| [5](#0705) | [6](#0706) | [7](#0707) | [8](#0708) | 9 | [10](#0710) | 11 |
| [12](#0712) | [13](#0713) | [14](#0714) | [15](#0715) | [16](#0716) | 17 | 18 |
| [19](#0719) | [20](#0720) | [21](#0721) | [22](#0722) | [23](#0723) | 24 | 25 |
| [26](#0726) | [27](#0727) | [28](#0728) | 29 | [30](#0730) | 31 | 1 |

#### August

| Mo | Tu | We | Th | Fr | Sa | Su |
| --- | --- | --- | --- | --- | --- | --- |
| [2](#0802) | [3](#0803) | [4](#0804) | [5](#0805) | 6 | 7 | 8 |
| [9](#0809) | 10 | 11 | 12 | 13 | 14 | 15 |
| 16 | 17 | [18](#0818) | [19](#0819) | 20 | 21 | 22 |
| [23](#0823) | 24 | 25 | 26 | 27 | 28 | 29 |
| [30](#0830) | [31](#0831) |  |  |  |  |  |

#### September

| Mo | Tu | We | Th | Fr | Sa | Su |
| --- | --- | --- | --- | --- | --- | --- |
|  |  | [1](#0901) | [2](#0902) | 3 | 4 | 5 |
| [6](#0906) | [7](#0907) | [8](#0908) | [9](#0909) | [10](#0910) | 11 | 12 |
| [13](#0913) | [14](#0914) | [15](#0915) | [16](#0916) | 17 | 18 | 19 |
| [20](#0920) | 21 | 22 | 23 | 24 | 25 | 26 |
| [27](#0927) | 28 | 29 | 30 |  |  |  |

## Notes

---

#### 06/28

First Day in the Lab!
Inoculation of BL21 strain and BL21-Omp deletion strain to 5ml LB buffer (3 each).
37 degree Celsius culturing overnight.

---

#### 06/30

**Redo the transformation for all six samples ([pSB1C3](http://parts.igem.org/Part%3ApSB1C3), pSB3C3, [pSB3K5](http://parts.igem.org/Part%3ApSB3K5), [pSB4A5](http://parts.igem.org/Part%3ApSB4A5), [pSB4C5](http://parts.igem.org/Part%3ApSB4C5), [pSB4K5](http://parts.igem.org/Part%3ApSB4K5)) following the [protocol](https://www.addgene.org/protocols/bacterial-transformation/)
Inoculation of colonies from a few strains' plates and preparation for the DNA extraction and genotyping.
(O)MVs harvesting**
OD600 measuring for six samples (diluted with water by 10 fold) (see Table 1).
Preparation of the solutions for centrifuge: If the weight of the liquid is not equal, add water (samples can vary maximally by 1g)
Centrifuge the sample: 8000 RMP at 4 degrees for 20 min --> gets rid of debris and some of DNA
Filtering with falcon pump the samples' solution and storing them in bottles. The samples treated with MMC showed a more sticky property and changing filtering paper is necessary. (Another option is using a vacuum filtering system). Make sure to wash the filter apparatus after every sample
Centrifuging the solution, removing the supernatant, and getting (O)MVs from the precipitate. The condition is 150,000x g, 4 ℃ for 1h. Make sure the samples are balanced(using LB buffer). And this step was done three times to obtain all (O)MVs.
After centrifuging, resuspend the precipitate by mili-Q water and centrifuge again in the same condition.
Discard the supernatant and resuspend the precipitate with different amounts of water according to the (O)MVs we obtained from step 4 (see table 2).
Storing the concentrated (O)MV sample in freezer.

![](https://static.igem.org/mediawiki/2021/6/6c/T--UZurich--notebook_0630_image_table_1_2.png)

![](https://static.igem.org/mediawiki/2021/8/8c/T--UZurich--notebook_0630_image_table_2_2.png)

![](https://static.igem.org/mediawiki/2021/5/54/T--UZurich--notebook_0630_image_3.png)

---

#### 07/01

**DNA extraction following the protocol: see Miniprep Protocol.**
 **Inoculation of colonies from all six plates(from yesterday's transformation) and prepare
for genotyping.**
 **DNA density measurement: using NanoDrop.**
The principle is to measure the light absorption from purine base and pyrimidine base which is around 260nm.
We sometimes can also see the peak from ethanol(~220nm) and from protein(~280nm)
First open the program and choose Nucleic Acid.
Adding water(1.5 µl) and do the initialisation
After initialisation using water to do the calibration(set the baseline)
Then using tissue to clean the detector.
Put 1.5 µl sample on the detector and start the measurement.
Record the concentration and A260/A280 ratio and clean the detector
Following the steps above and finish all the measurements.
Result is shown in table 1.

![](https://static.igem.org/mediawiki/2021/7/7b/T--UZurich--notebook_0701_image_1.png)

---

#### 07/02

**Plans:**
(O)MVs quantification
Miniprep the inoculated samples from 01.07(6 samples)
Restriction enzyme treatment and run the gel to separate the linear backbone
If we have microsynth's order we can do further PCR reaction.
(O)MVs quantification
**Protein quantification: using [BCA protein quantification kit](https://www.thermofisher.com/order/catalog/product/23227#/23227)**
Preparation of standard calibration with BSA: 2000µg/ml, 1000µg/ml, 500, 250, 125, 62.5, 31.25, 15.625, etc.(details can be found in excel file later) --> have a known absorbance curve (give us the linear curve in image 1, data points represent the absorbance of our own samples)
Mix the reagent A and reagent B(A:B = 50:1).
Preparation of sample: 1µl sample + 9 µl water + 10 µl 4% SDS + 180 µl reagent mix (reagent mix is added last!). (Depending on the concentration of our sample we can use different ratio of sample and water but in total 10µl)(for calibration we add 10 ul sample without water)
Incubation of the sample for 15-30min in 37℃ (watch the color change)
Do the quantification at absorption of 562nm.
Using the calibration data to do the linear regression.
Fit the sample data to this model and calculate the protein concentration.
Result is shown in table 2 and image 1.
**Lipid level test: using FM™ 1-43**
Dilute the FM 1-43 in 1:1000. (we mix 0.75 µl FM with 750 µl of water to obtain 1:1000)
Mix 1 µl sample with 100 µl of diluted FM 1-43. (The blank is made by only 100 µl of diluted FM 1-43)
And Quantify directly with exctiation in 485nm and emition in 590nm.
Result is shown in table 1.

![](https://static.igem.org/mediawiki/2021/b/ba/T--UZurich--notebook_0702_image_table_1.png)

![](https://static.igem.org/mediawiki/2021/9/90/T--UZurich--notebook_0702_image_table_2.png)

![](https://static.igem.org/mediawiki/2021/6/60/T--UZurich--notebook_0702_image_table_3.png)

*Seedling growth inhibition with isolated vesicles*

Question: What response do we get from the seedlings when treated with hight concentration vesicle treatment? Which treatment results in an ERF response in the seedlings?
Using 2 x 48 well plates
We left out the BL21 treatment for now
Using two strains of plants (efr-1, col-1)
Use MS as medium
Normalise vesicle samples by protein
End concentration of vesicles per well: 5 μg / 1 ml
Per well we fill in 500 μl
Volume of treatment solution: 5 μg/value from table 2 (μg/ml) --> gives the amount of ml of treatment per well (transform into μl by multiplying with 1000)
Dilute the vesicle samples (we had very small quantities of vesicles) 1:10 = vesicles:water --> 50 μl of vesicles and 450 μl of water (total 500 μl per well)

![](https://static.igem.org/mediawiki/2021/e/ee/T--UZurich--0702_image_table_4.png)

Handling seedling:
Desinfect workspace and hands
Use MS as base solution
Mix a tube with MS and amount above of treatment solution (don't forget to pipette out the same amount of MS out of the tube as is added from the treatment to prevent spilling)
Pipette 500 μl of the mix into the wells
Add seadling into every well
Seal the lid with breathable tape
Store in seedling room

Plasmid quantification from new samples:
Use 1.5 μl of water to apply onto the machine (name?)
Click on blank, then measure --> make sure output is about 0
Use 1.5 μl of the sample and note 260/280 ratio (protein to DNA ratio) and purity of the sample

Results

![](https://static.igem.org/mediawiki/2021/0/0e/T--UZurich--notebook_0702_image_table_5.png)

---

#### 07/05

**Plan:**
Most likely we will spend all time on molecular cloning.
Purify the pSB backbone(Gel purification)
Making our pGGA' and pGGE' plasmid(Using primer: iGEM1\_pGGA1\_R(F) and iGEM1\_pGGE1\_R(F) for backbone and iGEM5\_T7-lacO\_F(R), iGEM7\_T7Ter\_F(R) as target)
Making dummy entry vectors
Starting Greengate reaction to anneal the entry vectors
During greengate we can do another reaction to insert clyA to the pSB backbone and if possible, start transformation.

**Work**
Digesting the backbone plasmids for Cly-A + Epitopes to get the linear backbone and cut out the useless fluorescence protein fragment.
Resuspend the dry DNA: ClyA, epitopes
Gel purification and DNA extraction for ClyA-Epitope backbones: pSB1C3, pSB3C5 and pSB4C5
Setting up a gel purification: pSB(1C3, 3C5 and 4C5) backbones (preparing the tray)
Unpacking the received synthesized DNA (Cly-A + Epitopes)
Following the [protocol](https://eu.idtdna.com/pages/education/decoded/article/tips-for-working-with-gblocks-gene-fragments) to prepare the dried DNA
We want to do a control gel purification with the undigested backbone (Cly A + Epitopes) aswell (so getting these aswell as the digested ones)
Preparing PCR tubes with all the backbones(for adding Cly A&Epitopes) for gel purification (20 mi/L 2x high copy number backbone (digested), 1x 5 mi/L high copy number backbone (undigested), 2x 20 mi/L medium copy number backbone (digested), 1x 5 mi/L medium copy number (undigested), 2x 20 mi/L low copy number backbone (digested), 1x 5 mi/L low copy number backbone undigested)
Setting up the gel electrophoresis in the Et-Br room
Adding loading dye (sample volume/ 5)
Adding DNA ladder 1000 bp
Loading the gel
Performing the gel extraction: using the knife to cut the band from the gel under the UV light and purification of the Cly-A + Epitopes gel according to the protocol on the E.Z.N.A. gel extraction kit (Nanodrop result see table 1)

![](https://static.igem.org/mediawiki/2021/f/f5/T--UZurich--notebook_0705_table_1.png)

PCR reaction for making pGGA' (pGGE') backbones: 32µl dH2O, 10µl Greenbuffer, 5µl dNTPs, 1µl iGEM1\_pGGA(E)1\_R, 1µl iGEM1\_pGGA(E)1\_F, 10ng of template and 1µl Polymerase.
Setting up another gel purification for the T7 Promoter/Terminator and RBS/Terminator backbone
Performing the gel extraction and purification of the T7 and RBS backbone gel according to the same protocol
Prepare the working solution of the primers and T7, T7T oligos in 1:10 dilution.
Making a mixture of T7(R and F, 10 mili liter each and 80 mili liter of water to get another 1:10 dilution), T7T (Same as in T7), and two dummies(same as in T7)
Putting the fragments for insertion into the PCR for annealing (Temperature slowly decreasing from 97.5 to 10 in steps of 2.5)

For pGGA & pGGE green gate we add (T7 Promoter or A' (Dummy) for pGGA and T7 Terminator or E' (Dummy) for pGGE), 8 miL of Fr 1 miL T4 Ligase, 1 miL BsaI, 1 miL T4 Ligase buffer, then fill up to either 10, 15 or 20 miL with water (whichever needs the least addition of water)
For the Cly-A Golden Gate we add (4 miL pSB1C3 or 3 miL pSB3C5 or 1 miL pSB4C5), 8 miL Cly-A fragments (annealed (ds)), 1 miL T4 Ligase, 1miL BpiI, 1.5 miL T4 Ligase buffer, then we fill up to either 10 miL, 15 miL or 20 miL with water (whichever needs the least addition of water)
For time-saving reasons additionally to producing the entry vectors in the PCR (takes 3h) we parallel produce the pSB1C3 plasmid with T7 Promoter, RBS, Venus (NLS), Venus, Venus, Terminator, T7 Terminator directly with the dsDNA and let it run overnight (less efficient).

---

#### 07/06

**Work**
Before lunch:
Making a gel (useless though...)
Take the PCR tubes and do the transformation (Four PCR tubes are used for the transformation):
The first one is pSB1C3 as backbone and T7, T7 terminator and pGGA-pGGE as entry "vectors"(we use T7 and T7 terminator in dsDNA form)
The second one is pSB1C3 as backbone and two dummies at the begin and the end and pGGA-pGGE as entry "vectors" (we use two dummies in dsDNA form)
The third one is pSB4C5 as backbone and T7, T7 terminator and pGGA-pGGE as entry "vectors"(we use T7 and T7 terminator in dsDNA form)
The last one is is pSB4C5 as backbone and two dummies at the begin and the end and pGGApGGE as entry "vectors" (we use two dummies in dsDNA form)
The transformation follows the [protocol](https://www.addgene.org/protocols/bacterial-transformation/?gclid=CjwKCAjwhuCKBhADEiwA1HegOaTXdwoR4ZnEUpLexjcpMoTMspuWLXlmlAP6YbwFxzBAUTKnkYqPVxoCUGYQAvD_BwE) and this time we use 7.5µl of DNA

After lunch:
Seed sterilisation: 3 eppendorf tubes each for efr-1 and col seeds and put them into the sterilisation chamber with 50 ml 5% bleach and hcl (this starts a reaction that forms chlorine gas) leave them for 4 hours
After transfer the seeds (dispersed pay attention that there are no clumps) onto MS plates, close them with breathable tape and put into cold room in the dark box for stratification (we can put them into the grow room thursday or monday and need to use them 4 days after that).
Also discard the eppendorf tubes into biowaste bc can be seeds stuck to the wall

Also we ordered soil plates and need to collect them on thursday (for the ROS assay)
Label the white label with iGem and put a yellow (bc arabidopsis-transgenic) in each of the pots stating the genotype (we need to mix the genotypes)
- finished the transformation and put onto 3 plates, 2 for 1C3 and one with T7 and blank for the 4C5
- redoing plates for clyA for pSB4C5 and pSB3C5 because they didnt grow (spreading 150 microliters this time)

---

#### 07/07

**Plan**
Redo series from 05.07 and 06.07 (received no results)
Do digestions
Do PCR and gel extraction/purification with pGGA/E and 1C3, 4C5, 3C5
Annealing ssDNA to dsDNA

**Gel extraction of pGGA'/E'**
Tube weights:
A': 0.4 g
E': 0.6 g
Follow OMEGA protocol in Gel Extraction Kit
Quantify the extracted DNA with nano drop:

![](https://static.igem.org/mediawiki/2021/6/66/T--UZurich--notebook_070_table_1.png)

**Gel extraction of 4C5, 3C5, 1C3**
Weight of tubes:
4C5: 0.1 g
3C5: 0.1 g
1C3: 0.1 g
Quantification

![](https://static.igem.org/mediawiki/2021/0/04/T--UZurich--notebook_0707_table_2.png)

Annealing
Use R and F primers from primerbox: Tubes 5-14
Dilute primers by 10 with water
Ratio small fragment:backbone = 3:1
Calculate concentration of annealed solution: We need to insert 1 ul dsDNA into pGGA'/E'

Starting the golden gate reaction
Starting liquid cultures with the 1C3 vectors at different antibiotic concentrations: 0.5, 1, and 5 miL

---

#### 07/08

**Work**
Doing the transformation of Cly-A into the 1C3, 4C5 & 3C5 backbones.
Doing the transformation of the T7 promoter and terminator and 2 dummies into the pGGA and pGGE backbones.
Doing a mini-prep for 1C3 cultures at different concentrations of antibiotics
We planted the seedlings into the soil and put them into ARALAB 18 (need to remove the lid tomorrow (09.07) will be ready in 3 weeks (29.07))
Prepared a 10 mL culture with K antibiotic of the strain: JW3132 nlp1 for making competent cells out of it tomorrow.
Measured concentrations of all the unaltered backbone stocks from iGEM (Only the C backbones) and picked the highest concentration for sequencing
We took the 1C3 5 miL liquid culture as well as the 4C5 and 3C5 strains and prepared it for sequencing
We filled out the in-house sequencing form and sent it to Lena Stransfeld (Order is 1C3 13, 3C5 13, 4C5 13, 1C3 14, 3C5 14, 4C5 14)

![](https://static.igem.org/mediawiki/2021/3/3a/T--UZurich--notebook_0708_image.png)

---

#### 07/10

**Work**
Made competent cell with the nlp-1 knock-out strain. Did 1/10 of the recipe.
Tested the competent cells via transformation with the 4C5 backbone. Results tomorrow. Added 1 microliter of DNA).
Ligated PGGA backbone to A' and T1 Promoter and the PGGE to the E' fragment and the T7 Ter
Transformed the result from the ligation with the normal competent cells.

---

#### 07/12

**Work**
Greengate reaction to build up a vector: backbone: pGGZ; entry vectors: pGGA-pGGF. (1 µl of each vector, 1 µl BsaI, 1 µl buffer and 1 µl water)
Do the transformation using this resulting vector to competent Ecoli cells.
Colony PCR of colonies from plate: pGGA' and pGGE'. Three colonies from each plate were chosen.
Run a gel of colony PCR result. Three bands were get from pGGA' colonies. After comparing with the theoretical result, we decided to do a sequencing to these colonies tomorrow and therefore we inoculate these three colonies.
Growth inhibition tests are done and we scaled the seedlings.
We decided to isolate OMVs from our strains(Lpp deleted strains) so we start with a small scale inoculation.
New primers and strategies are designed for fusion-detection vectors.

![](https://static.igem.org/mediawiki/2021/f/f3/T--UZurich--notebook_0712_image_1.png)

![](https://static.igem.org/mediawiki/2021/6/6d/T--UZurich--notebook_image_2.png)

![](https://static.igem.org/mediawiki/2021/f/fa/T--UZurich--notebook_0712_image_3.png)

![](https://static.igem.org/mediawiki/2021/6/65/T--UZurich--notebook_0712_image_4.png)

![](https://static.igem.org/mediawiki/2021/a/a8/T--UZurich--notebook_0712_image_5.png)

---

#### 07/13

**Work**
Extract plasmids from yesterday's pGGA' inoculation and do a squencing.
New primers are designed and purchased for synthesis.
Briefl analyzed the data from the seedlings growth inhibition test.
Run the growth curve of the strain: BL21, Omp, NlpI, rseA, degP, TolA and TolB using yesterday's culturing.
Re-inoculate these seven strains for tomorrows OMV isolation.

---

#### 07/14

**Work**
Analysis of data from growth curve experiment.
Plasmid isolation of pGG: pGGZ as backbone and pGGA-F as entry vector.
pGG plasmid digestion: using 4 µl plasmid, 1 µl KpnI, 1 µl XbaI, 3 µl 10 x buffer(with BSA) and 21 µl water. After 2h digestion run the gel. Result will be uploaded. (Plan to use primers to do the PCR and get the middle pGGA-F part for justification)
Preparation of glycerol stock bacteria strains: BL21, Omp, NlpI, rseA, degP, TolA and TolB
Test the OD 600 of the strains we want to use for tomorrow's OMV isolation and inoculated them to a 200ml culture erlenmeyer. The OD 600 for each strains will be uploaded later.
**Plasmid isolation: pGGZ as backbone, pGGA-F as small fragment**
Nanodrop readout:

![](https://static.igem.org/mediawiki/2021/7/74/T--UZurich--notebook_0714_image_1.png)

Digest the plasmids into a linear strand (with Kpn1 and Xba1) and run through electrophoresis
Verdict: The golden gate method is suitable to build the designer plasmids (verify with Philipp)

**Create new OMV's with new strains**
OD600 of the following strains in small culture
Dilute to 0.01 OD600 in 200 ml LB: 200x0.01/OD600 = ml
Attention: We diluted to 0.02 OD in 200 LB for our 200 ml culture

![](https://static.igem.org/mediawiki/2021/d/df/T--UZurich--notebook_0714_image_2.png)

Transfer the diluted amount of small culture bacteria into a 200 ml culture of LB and let grow overnight

---

#### 07/15

**Plans**
Isolate OMV from the new stains: omp8, degP, RseA, TolB, TolA, Nlp1
Following workflow from the 30.06.21 Lab Journal

**Work**
Optical density of the 200 ml bacteria culture [OD600]:
Omp: 3.77
Nlp1: 5.69
RseA: 5.08
deg P: 4.37
Tol A: 6.17
Tol B: 4.46

Isolation
Samples were centrifuges three times and resuspended, then frozen
Tol A and resA didn't show a pellet

---

#### 07/16

**Plans**
Wash and centrifuge and resuspend the OMV samples from yesterday
Do lipid and protein quantification for the new OMV samples
Do lipid quantification for the old OMV samples (last time we only did protein quantification)

**Work**
**OMV sample preperation**
Resuspend the pellets in following volumes:
Nlp1: 200ul
Omp8: 200ul
TolB: 200ul
deg P: 50ul
rseA: 230ul
TolA: 160ul

**Protein quantification**
Prepare reagent mix: 3000ul of reagent A and 60ul of reagent B (total volume: 3060ul)
Add 1 ul of sample and 9ul of water (in total 10 ul), add 10ul of 4% SDS
Add 180ul of reagent mix
Tip: If bubbles emerge, poke them with a tip
Incubate for 15 to 30 min
(more details: see Lab Journal 02.07.21)

**Lipid quantification**
Use column 5 of the welled plate
Dilute the FM 1-43 in 1:1000. (we mix 0.75 µl FM with 750 µl of water to obtain 1:1000)
Mix 1 µl sample with 100 µl of diluted FM 1-43. (The blank is made by only 100 µl of diluted FM 1-43)(for sample C and F we add 5ul instead of 1)
And quantify directly with exctiation in 485nm and emition in 590nm.

**Make lipid normalisation for the old OMV samples**
Use Omp sample for normalisation
Dilute the following samples by 10 (some are already diluted, some need to be diluted again):
PAO1 MMC, BL21 MMC, omp, omp MMC
Volumes of 10 fold diluted samples added to the MS medium:
Pao1 MMC: 199.3ul
BL21 MMC: 425.7ul
omp: 199ul
omp MMC: 458.9ul

**Make dilution series for the new samples**
For a selected sample (we selected omp) we begin with 50ul and dilute in steps of

---

#### 07/19

**Plans**
Looking for viral vectors on the internet
Culturing different bacterial strains for comparison of MV production and fusion:
Agrobacterium tumefaciens grows at 28°C optimally
Pseudomonas putida IsoF (leo eberl) grows at 30°C optimally
Pseudomonas syringae grows at 28°C optimally
Pseudomonas fluorescens CHAO (leo eberl) grows at 30°C optimally
Bacillus subtilis 168wt (PxylA-xhlAB-xlyA) (leo eberl) grows at 37°C optimally
Preparing cultures for competent cells BL21 omp8 and TolB and BL21 wt

**Work**
Prepared the culture of BL21 omp8 for competent cells, made two 5 ml liquid cultures (one from old BL21 omp8 plate and one from the new one) put it into the shaker at 14.30
Prepared 6 seedlings (3x efr & 3x col0) and put them into the stratification box

---

#### 07/20

**Plans**
Create competent cells from over night culture
Molecular cloning if primers arrive
Receiving shop card from Lena
**Work**
Preparing compentent cells of BL21 wt, BL21 omp8 & TolB (following the protocol of the lab)
We got the shop card
Getting the bacterial strains from Leo Eberls Lab stocks on our prepared salt-free agar plates (P.Putida, P. Fluorescence and B. Subtilis) (Will arrive next morning)
Preparing agar plates with Rifampin (Rif) & Gentamicin (Gent) for the strain A. tumefaciens and Rifampin (Rif) & Kanamycin (Kan) for the strain P. Syringae (We will get both strains from Kyle)
Bought stuff at the lab
We need to order the following:
[Pierce™ BCA Protein Assay Kit](https://www.thermofisher.com/order/catalog/product/23227#/23227)
[Durapore® Membranfilter, 0,45 µm](https://www.sigmaaldrich.com/CH/de/product/mm/hvlp04700)
Received the primers from Microsynth
Started with the molecular cloning
Following the PCR protocol:

![](https://static.igem.org/mediawiki/2021/8/8d/T--UZurich--notebook_0720_image_1.png)

Prepared a gel and performed gel electrophoresis of the PCR product.

![](https://static.igem.org/mediawiki/2021/c/cc/T--UZurich--notebook_0720_image_2.png)

iGem 18 & iGem 19 together with 1C3 or 4C5 did not work
Gel extraction of the ones that did work
Redoing the PCR for the faulty ones (iGem18 with 1C3 and iGem 19 with 4C5 and iGem 22 & 23 with 3C5)
To one of the new PCR products (6) we added the restriction enzyme DPN1 to cut the methylated backbone (which is not a PCR product)

---

#### 07/21

**Plan**
Gel preparation for 3 & 4 which did not work (now with adjusted Tm)
Transformation of 1C3 & 4C5 + Cly-A
For PCR Nr. 6 which is iGem 22 and iGem 23 + 3C5 of the gel extracted DNA of the first
PCR from yesterday and with the new one which is digested to clean it from residues
Getting the bacterial strains (P.Putida, P. fluorescence and B. subtilis from Leo Eberls lab)
Creating a bacterial stock for these strains

**Work**
Gel preparation and gel electrophoresis (Let it run for 30min)
Transformation of 1C3 & 4C5 + Cly-A
Getting the bacterial strains from Leo Eberls lab
Gel extraction of PCR products 3 & 4 following the [protocol](https://ch.promega.com/-/media/files/resources/protcards/wizard-sv-gel-and-pcr-clean-up-system-quick-protocol.pdf?rev=29aa4610719c4f96afd213c345bc76d3&sc_lang=en)
The concentrations are 41..5 ng/miL and unknown purity for PCR product 4 and 25.8 ng/miL and purity 2.02 for PCR product 3

---

#### 07/22

**Work**
We got colonies from the transformations we did yesterday exept for 4C5 Cly A with the normal comp cells and 6 gel extracted one
Colony PCR on the Cly A constructs so 1C3 cly A Tol B , 4C5 Cly A Tol-B and 1C3 Cly A with normal comp cells
Vol 25 mil 16 h2o 5 green buffer 2.5 dNTP 0.5 VF2, 0.5 VR o,5 X7 Polymerase
Transformation of the Green gate constructs songyang did yesterday
1C3 PT7 35S omega enhancer NLS 3x Venus hsp18 T7Ter
1C3 PT7 ClyA Flag 22
4C5 PT7 Cly a elf18
4C5 PT7 35S omega enhancer NLS 3x Venus hsp18 ter T7ter
Made new Cm plates
redid Rif+Kan plates bc p. syringae didnt grow so tryed again is now in the 28 incubater
Made Glycerol stocks of B.Subtilis, p. fluirescence CHAO and p. putida
Made a gel withe the colony PCR product
Inoculated 3 replicates of the 6 with dnp I and the without Dnp 1 (overnight 5ml culture in the shaker)
Inoculated agrobacterum strain 5 ml overnight culture to make glycerol stock tomorrow

---

#### 07/23

**Plans**
Prepare A. tumefaciens glycerol stock
Order soil
Put 1 plate of each seed in to the grow room to put in soil on Tuesday

**Work**
Did miniprep on our overnight cultures
Results yield purity
4C5 Cly A -1 169.1 1.79
4C5 Cly A -2 148.2 1.78
4C5 Cly A -3 153.2 1.66
1C3 ClyA -3 109.6 1.72
1C3 ClyA -2 165.2 1.63
1C3 ClyA -1 65.0 1.72
1C3 35S 1 15.4 1.47
1C3 35S 2 74.4 1.76
1C3 35S 3 211.4 1.82
3C5 Bsa1 Mut -1 367.2 1.84
3C5 Bsa1 Mut -2 241.1 1.81
3C5 Bsa1 Mut -3 96.6 1.71
3C5 Bsa1 MutDpn1 -1 243.5 181
3C5 Bsa1 Mut Dpn1-2 219.3 1.81
3C5 Bsa1 Mut Dpn1 -3 186.6 1.82

Testing these:
For clyA constructs via PCR
React. vol. 25 mil (16 h2o, 5 green buffer, 2,5 dNTP, o.5 VF 2, 0.5 VR, 0.5 X7 Polymerase
For the other two via digestion
For 35S: react vol 10 mil (2 mil plasmid (over 200 ng), 1 mil Spe1, 1 mil Xba1, 1 mil cutsmart Buffer, H2O)
For the BsaI constructs (IGem 6):
React vol. 10 mil. (over 200 ng plasmid, 1 mil Bsa1, 1 mil T4 Ligase buffer, H2O)
After run gel to see if they are correct
Put 1 plate of Col-0 and efr-1 seeds into grow room
Made agrobact. glycerol stock
Doing transformation with Tol-B comp cells with golden gate constructs from yesterday
(1C3 PT7 35S omega enhancer NLS 3x Venus hsp18 T7Ter
1C3 PT7 ClyA Flag 22
4C5 PT7 Cly a elf18
4C5 PT7 35S omega enhancer NLS 3x Venus hsp18 ter T7ter)
Ordered two soil trays and booked the aralab 18 for them
Running the gel of the digestion
Running the gel from the PCR (if bands look right will prepare glycerol stocks of the overnight liquid cultures)
Inoculated the colonies from plates:
(1C3 PT7 35S omega enhancer NLS 3x Venus hsp18 T7Ter
1C3 PT7 ClyA Flag 22)

---

#### 07/26

**Plans**
Evaluate seedling growth inhibition
Maybe start some OMV isolation

**Work**
Did miniprep on the 5 ml cultures of 1C3 T7 35S 4-8 and the 3C5 gel extraction Bsa1 mutant.
Results: yield purity (260/280)
gel+Dpn1 3C5 Bsa1 mut 163.2 1.88
1C3 T7 35S 4 126.0 1.76
1C3 T7 35S -5 94.1 1.70
1C3 T7 35S -6 134.7 1.82
1C3 T7 35S -7 48.4 1.75 29.0 1.68 32.3 1.84
1C3 T7 35S -8 47.1 1.68 100.4 1.53
For the 7 and 8 re did the elution and left them on the 60 degrees for couple minutes to evaporate the ethanol before centrifuging again.
Started 5 ml liquid culture of p. syringae with gent and rif to make glycerol stock later
Ran the gels for the miniprep products and other BsaI mut from last week for BsaI bands seem to be correct but we need sequencing to be sure. For the others gel looks wrong so we redid the green gate reaction.
Also we did seedling weighing for the seedling growth inhibition.

---

#### 07/27

**Work**
Sequencing:
Inoculation of strains for OMV and MV harvesting: Omp8, P.fluorescence, P.putida, P.syringae, agarobacteria, B.subtilis.
Mainly discussion.

---

#### 07/28

**Plans**
Meeting with C.Zipfel
Transfering the seedlings (Efr-1 & Col-0) into soil
Preparing bigger cultures for OMV isolation of the different strains (A. tumefaciens, P.syringae, P. putida, P. fluorescence CHAO, B. subtilis 618, E. coli BL21 omp8)
Redo the construction of the linearized backbone with T7 promoter and T7 terminator and redo the Green Gate reaction to create the fusion testing construct

**Work**
Meeting with C.Zipfel
Transfering the seedlings (Efr-1, Col-0) to soil
Received more seeds from Kyle (Col-0, Efr-1, Fls-2 & Efr-1 & Cerk-1 (tripple-mutant called fec))
PCR of the linearized backbone with T7 promoter and T7 terminator
Measuring OD600 of our bacterial strain liquid cultures and calculating the amount that we need to add to the 2 x 200ml flasks (one for OMVs and one for CMVs):
Formula: (Volume of target flask \* desired OD600)/Measured OD600
BL21 omp 8 new plate: 6.28 => transfer 0.64 ml into 200ml flask to reach OD600 of 0.02
BL21 omp 8 original plate: 0.98 => transfer 10.2ml into 200 ml flask to reach OD600 of 0.05
BL21 omp 8 glycerol stock: 3.21 => transfer 1.25ml into 200ml flask to reach OD600 of 0.02
P. flouresence: 1.51 => transfer 2.65ml into 200ml flask to reach OD600 of 0.02 (we only have 5ml so we take 2 times 2.5ml)
P. syringae: 0.33 => transfer 30.3ml into 200ml flask to reach OD600 of 0.05 (we only have 5ml so we take 2 times 2.5ml)
P. putida: 1.18 => transfer 3.39ml into 200ml flask to reach OD600 of 0.02 (we only have 5ml so we take 2 times 2.5ml)
A. tumefaciens: 4.78 => transfer 0.84ml into 200ml flask to reach OD600 of 0.02
B. subtilis: 2.85 => transfer 1.4ml into 200ml flask to reach OD600 of 0.02
Let 200 ml culture sit for 3 hours
After 3 hours add MMC to all the samples(except for p.syringae since the concentration is too low)
Doing the gel extraction of the linearized plasmid: Concentration later
Phosphorylating the ends of the linearized plasmid to make it circular(Using T4PNK, detailed amount later)
Run another Greengate reaction using linear 1C3 backbone and other 5 entry vectors.
Transformation of pGGC to amplify the plasmid

---

#### 07/30

**Plan**
Miniprep overnight cultures
Make Green Gate and transformation with overnight culture
Isolate OMV's

**Work**
Morning:
Zetrifuge OMV and CMV samples
Miniprep overnightculture via protocol provided by promega

Afternoon:
**Protein quantification**
Prepare reagent mix: 3000ul of reagent A and 60ul of reagent B (total volume: 3060ul)
Add 1 ul of sample and 9ul of water (in tottal 10 ul), add 10ul of 4% SDS
Add 180ul of reagent mix
Tip: If bubbles emerge, poke them with a tip
Incubate for 15 to 30 min
(more details: see Lab Journal 02.07.21)

**Lipid quantification**
Use column 5 of the welled plate
Dilute the FM 1-43 in 1:1000. (we mix 0.75 µl FM with 750 µl of water to obtain 1:1000)
Mix 1 µl sample with 100 µl of diluted FM 1-43. (The blank is made by only 100 µl of diluted FM 1-43)(for sample C and F we add 5ul instead of 1)
And quantify directly with exctiation in 485nm and emition in 590nm.

**Miniprep**
Nanodrop results:

![](https://static.igem.org/mediawiki/2021/0/09/T--UZurich--notebook_0730_image_1.png)

---

#### 08/02

**Plans**
Miniprep of 1C3 T7 mVenus construct in normal DH10 cells
Digestion for analysis of the construct using Xba1 & Spe1 restriction enzymes
Gel electrophoresis for testing if we see two bands
Reading the [article](https://www.frontiersin.org/articles/10.3389/fpls.2020.00253/full)

**Work**
Did the miniprep of the 1C3 T7 mVenus

Did the digestion for analysis
1: 35.7, 1.80/25.4, 1.69
2: 100.3, 1.73
3: 111.1, 1.79
4: 96.3,1.82
5: 76.6, 1.79
6: 90.8, 1.81
7: 124.2, 1.76
PGGD: 176.4, 1.80

Doing the Gel electrophoresis
Gel had no EtBr in it, so redoing the digestion
Preparing chemicals for a ROS assay
Getting tobacco plants for ROS assay
Doing an HR test on N.benth
1 = P.fluorescence CMV high
2 = P.fluorescence OMV high
3 = A.tumefaciens CMV high
4 = A.tumefaciens OMV high
5 = P.fluorescence CMV 1/10 diluted
6 = P.fluorescence OMV 1/10 diluted
7 = A.tumefaciens CMV 1/10 diluted
8 = A.tumefaciens OMV 1/10 diluted

Results:

![](https://static.igem.org/mediawiki/2021/6/6a/T--UZurich--notebook_0802_image_1.png)

![](https://static.igem.org/mediawiki/2021/3/34/T--UZurich--notebook_0802_image_2.png)

Following the CZL protocol for ROS assay
Doing a golden gate to ligate the 1C3 | 4C5 backbone and the Cly-A

---

#### 08/03

**Plans**
Finishing the ROS assay

**Work**
Reading the [paper](https://www.frontiersin.org/articles/10.3389/fpls.2020.00253/full)
Preparing the ROS solution:
- 2ml MilliQ H2O per row
- 2miL HRP
- 2miL luminol | LO/12
- decide on concentration of OMV/CMV add 50 or 100 miL (same volume per MV)
- add everything to small falcon tubes
Doing the ROS with N. benthamiana and P.fluoresence OMV | CMV, A. tumefaciens OMV | CMV, E. coli BL21 omp8 OMV | CMV, Positive control flg-22, negative control H2O
- To reach a concentration of 10 mig/ml add:
- P.flourescence OMV 180 mil
- P.fluorescence CMV 18.3 mil
- A.tumefaciens OMV 371 mil
- A.tumefaciens CMV 27 mil
- E.coli BL21 omp8 OMV 52 mil
- E.coli BL21 omp8 CMV 27 mil
Sadly the ROS did not work (flg-22 response was too low) probably because N. benthamiana was rubbish
Moffat repeated the ROS in lettuce with our P.fluorescence CMVs and it showed a very strong response
Preparing new seedlings A. thaliana col-0 | efr-1 | efr-1 & fls-2 & sec-1 tripple mutant | bak-1/ 5
Getting leaf discs of Moffats lettuce for another ROS assay for tomorrow
Doing Miniprep of 1C3 bpi 1 -2 (1 ml DNA)| 1C3 Bpi 1-1 (3ml DNA) | pGGB (1 ml DNA):
- Bpi1-1: 76.6 ng/ul, 1.81
- Bpi1-2: 22.4 ng/ul, 1.96
- pGGB: 78.9 ng/ul, 1.90
Doing the golden gate for the different backbones from above with Cly-A
Preparing 1C3 T7 mVenus 2-5 and 1C3 T7 PNK 1 & 3 for sequencing

---

#### 08/04

**Plans**
Do another ROS assay with P.fluorescence CMV, A.tumefaciens CMV | OMV, E.coli BL21 omp8 CMV|OMV, P.putida OMV
Meeting with Beat Keller to discuss HIGS/RNAi application to our project

**Work**
Did another ROS assay with P.fluorescence CMV, A.tumefaciens CMV | OMV, E.coli BL21 omp8 CMV|OMV, P.putida OMV
Meeting with Beat Keller
[Results](https://2021.igem.org/File%3AT--UZurich--notebook_0804_results_correct.xlsx)
Doing the miniprep from yesterday again (1C3 bpi 1 -2 (1 ml DNA)| 1C3 Bpi 1-1 (3ml DNA) | pGGB)
Nanodrop results:
- pGGB mVenus 268.6 ng/μl 1.66
- pGGD mVenus 175.7 ng/μl 1.85
- 4C5 Cly-A J23119 280.5 ng/μl 1.85
Colony PCR
Prepare 5ml overnight cultures of P.fluorescence, P.putida , P.syringae and E.coli omp8 for OMV harvest

---

#### 08/05

**Plans**
Start OMV/CMV isolation
Sequencing results of the GreenGate (Fusion reporter construct) are bad
Doing the GreenGate again, but screening many more colonies (80-100 instead of 8) Moffat advised us
Preparing P. syringae glycerol stocks
Using E. coli BL21 omp8 competent cells for the Cly-A construct (maybe that works better)
Our T7 Promoter - Backbone - T7 Terminator construct didn't work as well (but we also didn't screen many)
- Alternative would be to use HBLC or Page purified primers
Meeting with Caua

**Work**
Doing a new round of GreenGate for the fusion reporter casette construct and screening more colonies
Meeting with Caua:
- Order sequences with IDT:
o Order 1: T7 Promoter - 35S - Ω-TMV - 3x mVenus - HSP-18 - T7 Terminater
o Order 2: ESAR system with GFP with TEV recognition site (flanked by restriction sites) and replaceable TEVp (flanked by restriction sites)
o Order 3: Cly-A construct with inducible promoter or weaker constitutive promoter
o Order 4: ESAR system combination of Tol-B repressible and TEVp inducible
Use Microsynth for DNA sequencing
- We need labels (Caua has many of them at Irchel)
- There is a box for sending it in (also at irchel)
- If we don't use PCR products, we need special tubes (need to order them in the future but Caua has some for now)
Started a PCR for the T7 Promoter-Backbone-T7 Terminater for sequencing with Microsynth
Prepared a glycerol stock of P. syringae
Decided on which OMVs/CMVs to isolate tomorrow (2x P. syringae CMV and OMV, 2x P. fluorescence OMV, 1x E. coli BL21 omp8 OMV and CMV)
Bringing PCR product to Caua for sending to sequencing

---

#### 08/09

**Plans**
Get omp-A plasmid
Do plasmid miniprep
SZ explain Cly-A construct
Check induced resistance
- Prepare P. Syringae
- MV treatment
IDT Order (first check with Caua)
Contact Microsynth because of repeated delays
3C5 mut seq
**Work**
Got omp-A plasmids
Did Miniprep
Contacted Microsynth because of delays: Delays due to quality control (they do it again if quality is bad and ship everything together)

---

#### 08/18

**Work**
Did an other infusion experiment with p. fluorescence CMVs and BL21 CMV to see what concentrations wont kill the cells
p. fluorescence start 7200 prot conc and dilute 10 times 3 times so 720 72 and 7,2
For BL21 start at 2800 to 280, 28 and 2,8
The highest concentration wasn't filtered
We use 4 N. benth plants and one leaf each with all the four treatments and one with 4 MQ infusions as negative control.
Also put seedlings into soil from bak 1-5, efr-1, fec and Col-O genotypes

---

#### 08/19

**Work**
Prepared the newly arrived primers and their dilutions.
Did miniprep on the new arrrived parts.
Nanodrop 19.08.21

---

#### 08/23

**Plans**
Weighing the seedlings for SGI
Doing another round of MV/OMV isolation
Preparing sequencing
Doing miniprep
Work
Weighed the seedlings for SGI
Did another round of OMV/MV isolation
Prepared the sequencing with Microsynth

---

#### 08/30

**Work**
Miniprep different constructs:
L1 tolB 1: 29.4/1.66
Flg elf 2: 65.9/1.72
L1 dCBD 2: 62.3/1.79
L1 TEV 2: 68.8/1.56
L1 TEV1: 17.7/1.58
L1 tolB 3: 36.4/1.89
Flg elf 3: 74.3/1.77
L1 dCB3: 67.7/1.87
L1 TEV 3: 23.9/1.69
L1 dCBD 1: 54.4/1.83
Flg elf 1: 135.0/1.71
L1 tolB 2: 52.4/1.72

Isolate OMV's from tolB constructs:

A1: tol B cly A elf18 1
B2: tol B cly A elf18 2
C3: tol B cly A flg22 1
D4: tol B cly A flg22 2
E5: tol B clyA only
F6: tol B clyA only no inducer

Get agrobacteria with plasmids from Prof. Voinnet:
- 35S:GF-FG in binary vector pBin61 obtained in Agrobacterium on Petri dish
- 35S: PVX210DIelta25 (GFP-tagged potato virus X minus movement and coat proteins) in binary vector pBin61 in obtained in Agrobacterium on Petri dish
- 35S: P19 in binary vector pBin61 obtained in Agrobacterium on Petri dish
Not forget that growth conditions differ for Agrobacterium (28°C, supplemented with rifampicin) and E.coli (37°C)

Innoculate HT115 (DE3) and L4440

---

#### 08/31

**Work**
Make competent cells from HT115 (see protocol CZL booklet)
Miniprep L4440
Plants are ready for ROS --> ROS done next week
Make SDS gel for western blot
Innoculate 3 agrobacteria strains with different vectors each (from Voinnet):
- 35S:GF-FG in binary vector pBin61
- 35S: PVX210Dlelta25 (GFP-tagged potato virus X minus movement and coat proteins) in binary vector pBin61
- 35S: P19 n binary vector pBin61

Sequence the following (three groups removed due to low concentration):
(L1 tolB 1) -> removed
Flg elf 2: 65.9/1.72
L1 dCBD 2: 62.3/1.79
L1 TEV 2: 68.8/1.56
(L1 TEV 1) -> removed
L1 tolB 3: 36.4/1.89
Flg elf 3: 74.3/1.77
L1 dCB3: 67.7/1.87
(L1 tev 3) -> removed
L1 dCBD 1: 54.4/1.83
Flg elf 1: 135.0/1.71
L1 tolB 2: 52.4/1.72

Sequencing (Micro Synth):
1: L1 dCBD 1
2: L1 dCBD 2
3: L1 dCBD 3
4: L1 TEV 2
5: L1 TolB 2
6: L1 TolB 3
7: flg elf 1
8: flg elf 2
9: flg elf 3
Miniprep L4440
Results Nanodrop:
L4440 1: 9

---

#### 09/01

**Plan**
Do western blot
Miniprep plasmid out of agrobacteria and transform them into DH10B:
35S:GF-FG in binary vector pBin61
35S: PVX210Dlelta25 (GFP-tagged potato virus X minus movement and coat proteins) in binary vector pBin61
35S: P19 n binary vector pBin61
Prepare leave discs from col-0 and erf-1 for ROS
Inoculate sequenced colonies from yesterday in 5ml culture:
Colonies: flg22-elf18-1, flg22-elf18-3, dCBD-1, dCBD-2, TEV-2, tolB-2, TolB-3

**Nanodrop Results from Miniprep**
35S:GF-FG in binary vector pBin61: **35.3 ng/ul** and 280/260 1.74
35S: P19 n binary vector pBin61: **66.3 ng/ul** and 280/260 1.70
35S: PVX210Dlelta25: **64.6 ng/ul** and 280/260 1.65

**Transformation of the plasmids we minipreped into DH10B**
Follow [Protocol](https://www.addgene.org/protocols/bacterial-transformation/)
Using: 2ul of plasmid

**Western blot**
Samples: TolB ClyA with inducer, TolB ClyA without inducer, TolB ClyA-flg22 with inducer, TolB ClyA-elf18 with inducer, omp8 Cly A with inducer, omp8 ClyA without inducer, omp8 ClyA-flg22 with inducer, omp8 ClyA-elf18 with inducer
Mix samples with loading dye

**ROS**
Make LO/12 stock solution
2mg of Lo/12 g in 6.4 ml MQ

---

#### 09/02

**Work**
ROS test using vesicles: tolB-ClyA-No inducer, tolB-ClyA, tolB-flg-1, tolB-flg-2, tolB-elf-1, tolB-elf-2
LMU goldengate: Para-ClyA-elf-flg, Para-ClyA-dCBD, Para-tolB, Para-TEV, Positive control and transformation
Transforamtion of BB05-BB09
Send sequencing

---

#### 09/06

**Work**
Miniprepped see below
Sent sequencing of the things we miniprepped beforehand

![](https://static.igem.org/mediawiki/2021/1/11/T--UZurich--notebook_0906_image_1.jpg)

Prepared the samples for the fusion test at the eth following their protocol

![](https://static.igem.org/mediawiki/2021/4/49/T--UZurich--notebook_0906_image_2.jpg)

![](https://static.igem.org/mediawiki/2021/a/af/T--UZurich--notebook_0906_image_3.jpg)

---

#### 09/07

**Work**
Prepared to redo the Ros assay with n. beth so we took leaf discs from 4 plants for one 96 well plate
Molecular cloning
First prepared gBlocks then set up the PCR reaction, see image below

![](https://static.igem.org/mediawiki/2021/a/a1/T--UZurich--notebook_0907_image_1.png)

Filtered and rediluted deltaTolB ClyA only induced CMVs for the ROS assay today we got a plate of Col-O to run from a friend who doesnt need it today, so we can run it.

![](https://static.igem.org/mediawiki/2021/6/6e/T--UZurich--notebook_0907_image_2.jpg)

---

#### 09/08

**Work**
Autoclaving bottles for OMV isolation
ROS test
Transformation of uTEV, eTEV and tolB with 3x TEV recognition site

---

#### 09/09

**Work**
OMV isolation of Cly-A uninduced, Cly-A induced, Cly-A elf-18 -1, Cly-A elf-18 -2, Cly-A, flg-22 -1, Cly-A flg-22 -2
PCR of GFP quencher for TevP testing
Gelextraction of the PCR product

---

#### 09/10

**Work**
Had meeting about the presentation video in the morning
Miniprepped and sent for sequencing following samples:

![](https://static.igem.org/mediawiki/2021/1/1a/T--UZurich--notebook_0910_image_1.png)

![](https://static.igem.org/mediawiki/2021/7/7f/T--UZurich--notebook_0910_image_2.png)

![](https://static.igem.org/mediawiki/2021/6/6e/T--UZurich--notebook_0910_image_3.png)

Went to the ETH to look at our infiltrated leaves with the confocal microscope (infiltrated with PVX and P19)
**We didn't see anything for the CMVs but for the agrobacterium (positive control) and for the *E.coli*!!**
We will discuss the results with professor Voinnet on tuesday at 2 pm but this may indicate that e. coli can transfer DNA into plant cells somehow or this is just an anomalie related to it being a viral vector.
Took leaf discs for ROS assay two plates of col-0 and one plate efr-1 to test the engineered vesicles again.

---

#### 09/13

**Work**
Miniprep of Para Cly-A elf-18 flg-22, dCBD
PCR
Gelelectrophoresis (Constructs incorrect except 1,2 and 8)
Restriction-Digestion test (All constructs incorrect)

---

#### 09/14

**Work**
Transformed uTEV, eTEV, tolB, tolB TEV, TEV into DH10 competent cells and inoculated them over night in the 37°C incubator
SGI with Lore, fec & col-0
Went to ETHZ to meet with Prof. Voinnet but he wasn't there.
Looked at the plants at ETHZ but it was too soon to see anything meaningful

---

#### 09/15

**Work**
SGI of plants treated with OMV's containing ClyA only, ClyA-flg22, ClyA-elf18
Colony PCR of uTEV, eTEV, tolB, tolB TEV, TEV
Subsequent gelelectrophoresis of the PCR product
Ladder, positive control, 5x TEV, 5x uTEV, 3x eTEV, Ladder
Ladder, positive control, 2x eTEV, 5x tolB, 5x tolB TEV, Ladder
Inoculation of the working strains (TEV, uTEV, eTEV, tolB, tolB TEV) in CARB LB
Inoculation of omp8 Cly-A only, omp8 Cly-A elf-18, omp8 flg-22 in CARB LB

---

#### 09/16

**Work**
Video meeting in the morning
Plan experiments with Prof. Voinnet after lunch
Miniprep: tolB TEV, tolB TEV 4, tolB 4, tolB 2, tolB 1, eTEV 5, eTEV 4, uTEV 4, uTEV 3, TEV 7, TEV 6
Make 200ml culture from omp8 ClyA, omp8 flg22, omp8 elf18
omp8 flg22: add 1.56 ml
omp8 elf18: add 1.08 ml
omp8 ClyA: add 0.99 ml

---

#### 09/20

**Work**
PCR of GFP-Quencher construct
Miniprep for workshop (failed)

---

#### 09/27

**Work**
Miniprep of workshop plasmids
Transformation of omp-A flg-22 elf-18 dCBD constructs

---

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### UZurich 2021

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# Team:UZurich/Engineering

* [iGEM UZurich 2021](https://2021.igem.org/Team%3AUZurich)
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* Project

  + [Description](https://2021.igem.org/Team%3AUZurich/Description)
  + [Simple Description](https://2021.igem.org/Team%3AUZurich/Simple_Description)
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  + [Proof of Concept](https://2021.igem.org/Team%3AUZurich/Proof_Of_Concept)
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* [Awards](https://2021.igem.org/Team%3AUZurich/Medal)

# Engineering Success

When a plant is confronted with pathogens, one of the first steps for the plant’s immune system is their recognition which consequently leads to the activation of several defence mechanisms in the plant to resist against the attacker. This is done in a process called PTI, short for pattern-triggered immunity. PTI is triggered when the plants pattern-recognition receptors (PRR’s) recognise conserved pathogen-associated molecular patterns (PAMPs). [1]

For Boom V, we decided to take advantage of this first innate immune response to prime the plants' immune system and therefore prepare them for a potential infection.

In the following text we are going to elaborate on our engineering cycle. Before we dive into the details, we will summarise the aims of each step of our engineering cycle:

1. Design: Expression of immunogenic epitopes such as flg22 ([BBa\_K3286138](http://parts.igem.org/Part%3ABBa_K3286138)) and elf18 ([BBa\_K3989023](http://parts.igem.org/Part%3ABBa_K3989023)) on the surface of bacterial outer membrane vesicles, which then can serve as PAMPs to PRRs
2. Build: Assemble and introduce the designed plasmid into a chassis where it can be expressed
3. Test: Conduct structural and functional tests to ensure our design can induce the PTI
4. Learn: Draw conclusions about the results and find ways to improve our product

![](https://static.igem.org/mediawiki/2021/c/ca/T--UZurich--engineering_cycle.png)
*Figure 1: iGEM engineering cycle*

### Our Cycles

* [Cycle 1](#Cycle1)

+ [Design](#Cycle1): Plasmid Design for ClyA construct
+ [Build](#Cycle1Build): Identification of Chassis

* [Cycle 2](#Cycle2)

+ [Design](#Cycle2): A different approach to plasmid design
+ [Build](#Cycle2Build): Cloning our construct
+ [Test](#Cycle2Test): Plant immunity assays and Western blots
+ [Learn](#Cycle2Learn): Some engineered OMVs trigger higher immune response

* [Cycle 3](#Cycle3)

+ [Design](#Cycle3): Using an iGEM backbone
+ [Build](#Cycle3): Improve the immune response
+ [Test](#Cycle3): Testing our constructs
+ [Learn](#Cycle3Learn): Our plasmid can be transformed into different chassis

## Cycle 1

### 1.1 Design

The purpose of our design is the delivery of the immunogenic epitopes to the surface of the bacterium, where they can be recognised by the plants PRRs. We chose a construct containing the gene for the ClyA protein ([BBa\_K811000](http://parts.igem.org/Part%3ABBa_K811000)), which after translation is integrated into the outer membrane. We linked genes for epitopes such as flg22 ([BBa\_K3286138](http://parts.igem.org/Part%3ABBa_K3286138)) or elf18 ([BBa\_K3989023](http://parts.igem.org/Part%3ABBa_K3989023)) to ClyA, which then places them on the surface of the bacterial membrane. In summary: We implemented ClyA to display epitopes and used the epitopes to trigger the pattern triggered immune response.

We based our construct on previous work done by Prof. Cyril Zipfel, which supports that PAMPs can trigger the PTI of plants [2] and other literature that shows that ClyA can be used as a displaying protein [3].

We wanted to achieve the above with a pre-synthesised plasmid containing following elements: ClyA-3xFLAG and the epitopes flg22 ([BBa\_K3989010](http://parts.igem.org/Part%3ABBa_K3989010)) or elf18 ([BBa\_K3989011](http://parts.igem.org/Part%3ABBa_K3989011)).

![](https://static.igem.org/mediawiki/2021/7/73/T--UZurich--engineering_Plasmid_1_epitope.jpeg)
*Figure 2: Plasmid containing ClyA construct controlled by a constitutive J23119 promoter ligated into a iGEM backbone*

### 1.2 Build

In a first step, we wanted to identify an appropriate chassis. Important was that the chassis would overproduce OMVs, which are buddings of the outer membrane of gram-negative bacteria. We chose an *E. coli* mutant with a deleted TolB gene for the proof of concept. The *tolB* strain lacks the periplasmic TolB protein which hence compromises the membrane integrity: The bacterial outer membrane and the inner cytoplasmic membrane become less attached. This leads to an increase in OMV secretion [4]. The *tolB* strain was chosen based on preliminary experiments that showed that both the OMV secretion rate (determined by quantifying the lipid content in the OMV sample) and growth rate were rather high compared to different deletion strains that overproduce OMVs.

![](https://static.igem.org/mediawiki/2021/a/ac/T--UZurich--lipnorm.png)
*Figure 3: OMV quantification based on lipid quantification of different E.coli strains*

We transformed the plasmid from above into the *tolB* mutant but were faced with difficulties with the transformation. During troubleshooting, we came up with a new design that would be more modular, which would bring us some important advantages over our current design (discussed in Cycle 2, Design). Therefore we decided to start a new design cycle.

## Cycle 2

### 2.1 Design

We decided to change our approach for the second design cycle. Instead of designing and synthesizing the plasmid as a whole, we designed fragments of the plasmid which could later be assembled via a Golden Gate reaction.

The improved design looks as follows: We used an LII backbone [5] and synthesized a gene fragment containing ClyA-3xFLAG, and we synthesized a gene fragment with the epitopes flg22 or elf18. We also used primers to add BsaI or BpiI restriction cutting sites to each end of every individual fragment. Then we performed a Golden Gate reaction to link the two fragments together. We first used a constitutive J23119 promoter ([BBa\_J23119](http://parts.igem.org/Part%3ABBa_J23119)) (more details in [Build](#Cycle2Build) section) then switched to a Para promotor (inducible by Arabinose) ([BBa\_I0500](http://parts.igem.org/Part%3ABBa_I0500)) to achieve an inducible epitope-displaying system. The FLAG tags are used for the Western blot, which can identify whether the construct is expressed in our chassis. The backbone of the plasmid is from our lab and can be changed by a digestion-ligation reaction.

The plasmid map is shown below:

![](https://static.igem.org/mediawiki/2021/8/8e/T--UZurich--engineering_Plasmid_2.png)
*Figure 4: Plasmid containing modular ClyA construct controlled by an inducible Para promoter ligated into an LII backbone*

This approach poses one key advantage to the previous approach: It is modular. The epitopes can be changed depending on what plant we want to treat (different plants have different preferences for PAMPs) and the ClyA can be exchanged too if one wanted to use a different outer membrane protein to display the epitopes. This allows us to easily adapt the construct to different experiments.

After synthesis we sequenced the plasmid to verify our Golden Gate reaction was successful. Later we would also test the functionality of the design in a series of experiments testing for the presence of the ClyA-epitope structure in our chassis as well as the immunogenicity of our construct (see [Test](#Cycle2Test)).

### 2.2 Build

The chassis in the second cycle was kept the same as in the first design cycle.
The construct described in the [Design](#Cycle2) section was built in fragments, which were assembled via Golden Gate. Thanks to the modular nature of our design we could assemble different variants of the original construct using Golden Gate cloning. For the experiment we had planned, we created variants with ClyA only without epitopes, ClyA with flg22, and ClyA with elf18.

While cloning our construct and its variants, which were under the control of a constitutive J23119 promoter, we ran into problems transforming the construct into our *E. coli*. In an attempt to fix the problem we decided to change the promoter. The reason for this decision was the fact that ClyA belongs to the α-PFT (pore-forming toxins) which can cause pore formation in the bacterial membrane and hence lead to cell death.[6]
Thanks to the Golden Gate cloning we could easily substitute the constitutive J23119 promoter with an arabinose inducible promoter (pBAD) without having to resynthesize the entire construct. This approach allowed us successful cloning and transformation. To verify that our builds are correct, we used colony PCR to get a selection of possible colonies containing our plasmid. We then sequenced the colonies that showed a plausible band on the gel. We proceeded to sequence the colonies that showed a positive result on the gel to be sure our plasmid is contained in the colony.

It must be noted that we are still unsure if the toxicity of ClyA was the true reason for the unsuccessful transformation of our construct with the constitutive J23119 promoter (previous studies showed that it can be used as a surface display tool without killing the cell [3]) or if the problem stemmed from unsuccessful cloning. The fact that we didn’t manage to grow colonies that we could have sequenced prohibits us from making any definite conclusions.

### 2.3 Test

For the testing of our system we used two different approaches. Firstly we wanted to make sure that the OMVs we harvested after inducing the ClyA construct with Arabinose truly contained the proteins by running a Western blot (structural confirmation). Secondly, we tested if the OMVs containing the epitopes could induce an immune response by conducting a seedling growth inhibition assay (SGI) and a reactive oxygen species assay (ROS) (functional test). The western blot, the SGI assay, and ROS assays all provide quantitative results while the western blot and SGI assay have absolute units and the ROS assay has relative units.
To analyze and represent our gathered data from the SGI assay and ROS assay, we relied on statistical tests and graphs created with R studio. We made a statistical test for each assay looking for a significant difference between the individual OMV treatments and the controls.

The Western blot functions as follows: When we run our engineered OMVs through the gel, we expect to detect a band for ClyA-epitope structure. The 3xFLAG we included in the plasmid serves as a binding site for antibodies.
In the western blot, if the OMVs contain the ClyA-3xFLAG-epitope structure they are supposed to express, we should see protein-specific bands. For OMVs containing a ClyA-3xFLAG-flg22, we expect a band at ~40kDa, for OMVs containing ClyA-3xFLAG-elf18 we expect about the same. These expectations were confirmed.

![](https://static.igem.org/mediawiki/2021/3/3a/T--UZurich--OMV_Westernblot.jpg)
*Figure 5: Graphical representation of the bands we received when running a Western Blot on *E.coli omp8* derived OMVs: ClyA-FLAG-flg22, ClyA-FLAG-elf18 and ClyA-FLAG all show a band at around 40 kDA which confirms that the ClyA construct was implemented in the OMVs*

The SGI is an assay that takes seedling weight as a measure for an immune response. We filled OMV solution (treatment) and MS medium (mock) into a well plate and placed the Arabidopsis (Col-0) seedlings into the wells. After ten days, the seedlings are weighed. The stronger the seedling growth has been stunted, the stronger the immune response was.
In the SGI assay, we expected to see a stunted growth of seedlings in OMV treated seedlings if the engineered OMVs induce an immune response. We used 20nM flg22 as positive control and MS medium as mock to which we compared our OMV results to. We used three types of OMVs as treatments: ClyA-flg22 OMVs, ClyA-elf18 OMVs, and Cly-A only OMVs. From the data gathered we can see that this expectation was met: We observe that for both ClyA-elf18 and ClyA-flg22 we can see a stronger immune response compared to ClyA without epitopes and the mock at concentration 20ug/ml.

![](https://static.igem.org/mediawiki/2021/2/20/T--UZurich--engineering_SGI_result.png)
*Figure 6: Plotted data from an SGI. We treated *A.thaliana (Col-0)* with our OMV variants ClyA only, ClyA-elf18 and ClyA-flg22. The ability of the ClyA-construct to reduce seedling weight is significantly influenced by the OMV variant we treated the plant with and the concentration of OMV variant used*

The ROS assay takes the amount of reactive oxygen species in a leaf disc as the measure for an immune response. For this assay, we punched leaf discs and let them sit in water overnight to make sure the immune reaction from the punch wound could subside. Then we placed the leaf discs into a well plate. We used water as a mock, flg22 at 200nM as positive control, and three treatments: ClyA-flg22 OMVs, ClyA-elf18 OMVs, and ClyA-only OMVs. The quantification of the ROS burst was done by a plate reader, which detects the luminescence of the ROS reacting with luminol that is generated in the leaf disc. This luminescence will be depicted as a peak (measured in RLU).
In the ROS assay, we expected to see a larger ROS burst in leaf discs treated with OMVs expressing epitopes compared to the mock (water) and the OMVs with a ClyA without any attached epitopes. This was truly the case with Arabidopsis (Col-0) leave discs treated with OMVs that expressed elf18 on their surface. The ROS burst is clearly higher in discs treated with ClyA-elf18 OMVs than in the leaf discs treated with the mock (water) or ClyA only OMVs. The reason why the ClyA-flg22 OMV didn’t induce a higher immune response is due to the fact that elf18 is more immunogenic than flg22 in Brassicaceae, to which our model organism belongs.
However it must be noted that the ClyA-elf18 expressing OMVs did not reach quite the levels of pure flg22. We could try and solve this problem by using a different membrane protein with more extracellular domains to display more epitopes at the surface of the vesicles (see [Learn](#Cycle2Learn)).

![](https://static.igem.org/mediawiki/2021/2/23/T--UZurich--engineering_ROS_over_time_tolB.png)
*Figure 7: Kinetics of ROS response in *A.thaliana (Col-0)*. As a treatment we used the *tolB (E.coli)* derived OMV variants ClyA-elf18, ClyA-flg22 and ClyA without epitopes (induced ClyA). Flg22 was used as a positive control, water as a mock and ClyA without epitopes as a negative control. We can see that ClyA-elf18 OMVs trigger a high ROS burst measured in RLU*

Since we didn’t use any metadata for our experiments, our experiments can be repeated by others as long as they have the plasmid used to express the epitopes. The individual parts of our construct, namely the epitopes and the promoter, can also be tested separately. Since we are using an inducible promoter (induced by arabinose), we used different concentrations of arabinose to test the expression level. This provides valuable data for other teams in the future. The epitopes we mainly tested for their immune triggering capability. Different epitopes elicit different immune responses in different plants, which can be quantified in immune assays. However, the outputs from the SGI and ROS assays are influenced by the environment and the plant, therefore the generated data may vary depending on the lab and location. Having said that, we will provide the R code for the analysis of the ROS assay data on the [Measurement page](https://2021.igem.org/Team%3AUZurich/Measurement#facilitate) which can be used by other teams.

### 2.4 Learn

From our data gathered from the SGI assay and the ROS assay we learned that at least some of our engineered OMVs do trigger a higher immune response compared to the controls and mocks. We could however try and increase the immune response further. We hypothesize that we could achieve this by using an *omp8* *E. coli* strain instead of the *tolB* strain. *omp8* expresses low Omp levels in the outer membrane which creates more surface area into which ClyA can be incorporated [7]. This would result in more epitopes being displayed on the bacterial surface and therefore also on the OMVs.

We also learned that we need to take into account what plants we wish to treat with our OMVs since different epitopes would be required to maximize the immune response due to different preferences for different PAMPs among plants. Thanks to the modular nature of our construct, this could be easily achieved.

## Cycle 3

### 3.1 Design

We used the same construct as in the design cycle 2, except that we transferred our construct into an iGEM backbone with lower expression levels than the backbone from cycle 2. Reason being is that a lower expression causes less disturbance to the bacteria cell membrane and therefore increases the viability of our bacteria.

### 3.2 Build

We changed our chassis for this cycle in an attempt to improve the immune response that our OMVs would elicit. Therefore we transformed our ClyA-epitope variants (ClyA only without epitopes, ClyA with flg22, and ClyA with elf18) from the second cycle into an Omp8 deleted *E. coli* strain (*omp8*). We again sequenced the colonies to verify that our plasmid had been taken up during the transformation.

### 3.3 Test

We tested the engineered *omp8* derived OMVs in a ROS assay. We had two questions in mind that we wanted to answer: Firstly we wanted to know if the ClyA-construct can elicit an immune response when they are transformed into the new chassis. Secondly, we wanted to know if the immunogenicity differs between engineered OMVs derived from *tolB* and *omp8*.

The answer to the first question is, that ClyA-elf18 OMVs and ClyA-flg22 OMVs both elicit an immune response in *A. thaliana* (Col-0), while the ClyA-elf18 OMVs again elicit a stronger immune response.

![](https://static.igem.org/mediawiki/2021/f/fb/T--UZurich--engineering_over_time_omp8.jpg)
*Figure 8: Kinetics of ROS response in *A.thaliana (Col-0)*. As a treatment we used the *omp8 (E.coli)* derived OMV variants ClyA-elf18, ClyA-flg22 and ClyA without epitopes (induced ClyA). Flg22 was used as a positive control, water as a mock and ClyA without epitopes as a negative control. We can see that ClyA-elf18 OMVs trigger a higher ROS burst measured in RLU compared to the mock*

As for the second question, we conducted a ROS assay with ClyA-only, ClyA-elf18, and ClyA-flg22 OMVs derived from *tolB* and *omp8* strains as treatments. The results show that our *omp8* derived ClyA-elf18 OMVs indeed manage to elicit a higher immune response compared to *tolB* derived ClyA-elf18 OMVs. With this result, we can confirm that a change in chassis from *tolB* to *omp8* does improve the immunogenicity of the OMVs.

![](https://static.igem.org/mediawiki/2021/archive/0/05/20211016220002%21T--UZurich--engineering_ROS_construct_tolB_omp8.jpg)
*Figure 9: Comparison of the induced ROS bust of *omp8* and *tolB* derived OMV variants ClyA-elf18, ClyA-flg22 and ClyA without epitopes (induced ClyA) in *A.thaliana (Col-0)*. We can see that the strain used significantly influences the ability of the OMV to elicit a ROS burst. Here all the *omp8* derived OMV (except for ClyA induced which is the construct with only ClyA and no epitopes) variants elicit the stronger ROS burst compared to the *tolB* derived counterparts*

### 3.4 Learn

From the experiments conducted in the [Test](#Cycle3) section we could learn that our plasmid can be transformed into different chassis and that by doing so, the OMVs don’t lose immunogenicity and can even become more immunogenic, if the chassis is chosen correctly.

These results provide necessary data that enables us to implement our design into a bigger picture: An inducible system. This inducible system would include our ClyA-construct that delivers epitopes to the surface and two other plasmids: A TEV-construct and a TolB-construct with a TEV cutting site. All three would be introduced into a *tolB* mutant strain of choice (e.g. a plant-associated soil bacterium such as *P. putida*), which lacks the TolB membrane protein that normally helps to hold the two membranes together. With this system, we would be able to produce OMVs in an inducible manner by both repressing the TolB gene in the TolB-construct and by activating the TEV protease gene with a quorum-sensing molecule. This would have the following as an effect: The protease will cut the produced TolB at the inserted TEV cutting site and additionally no new TolB is produced since the gene is repressed. For a more detailed description of this inducible system, go to  [Design](https://2021.igem.org/Team%3AUZurich/Design). If implemented in a plant-associated soil bacterium such as *P. putida*, the engineered bacteria could be released into a field where they could sense pathogens attacking the plant via their quorum-sensing molecules and secrete the immunogenic OMVs as a response to the attack.

For complete scientific names of the strains used see: [list strains](https://static.igem.org/mediawiki/2021/4/45/T--UZurich--list_strains_genotypes.pdf).

### [**go to top**](#protocols-image)

---

[1] Bigeard, J., Colcombet, J. and Hirt, H., 2015. *Signaling mechanisms in pattern-triggered immunity (PTI).* Molecular Plant, 8(4), pp.521-539.
[2] Monaghan, J., & Zipfel, C. (2012). *Plant pattern recognition receptor complexes at the plasma membrane.* Current opinion in plant biology, 15(4), 349–357.
[3] Kim, J. Y., Doody, A. M., Chen, D. J., Cremona, G. H., Shuler, M. L., Putnam, D., & DeLisa, M. P. (2008). *Engineered bacterial outer membrane vesicles with enhanced functionality.* Journal of molecular biology, 380(1), 51-66.
[4] Turner, L., Praszkier, J., Hutton, M.L., Steer, D., Ramm, G., Kaparakis‐Liaskos, M. and Ferrero, R.L., 2015. *Increased outer membrane vesicle formation in a Helicobacter pylori tolB mutant*. Helicobacter, 20(4), pp.269-283.
[5] Chiasson, D., Giménez-Oya, V., Bircheneder, M., Bachmaier, S., Studtrucker, T., Ryan, J., ... & Parniske, M. (2019). *A unified multi-kingdom Golden Gate cloning platform.* Scientific reports, 9(1), 1-12
[6] *[Pore-Forming Toxin](https://www.sciencedirect.com/topics/agricultural-and-biological-sciences/pore-forming-toxin)*
[7] Thoma, J., Manioglu, S., Kalbermatter, D., Bosshart, P.D., Fotiadis, D. and Müller, D.J., 2018. *Protein-enriched outer membrane vesicles as a native platform for outer membrane protein studies.* Communications Biology, 1(1), pp.1-9. (Figure 1a)

[![](https://static.igem.org/mediawiki/2021/8/8b/HQ_page_logo.jpg)](https://2021.igem.org)

# Team:UZurich/Experiments

* [iGEM UZurich 2021](https://2021.igem.org/Team%3AUZurich)
* |
* Project

  + [Description](https://2021.igem.org/Team%3AUZurich/Description)
  + [Simple Description](https://2021.igem.org/Team%3AUZurich/Simple_Description)
  + [Design](https://2021.igem.org/Team%3AUZurich/Design)
  + [Results](https://2021.igem.org/Team%3AUZurich/Results)
  + [Proof of Concept](https://2021.igem.org/Team%3AUZurich/Proof_Of_Concept)
  + [Best Plant Synthetic Biology](https://2021.igem.org/Team%3AUZurich/Plant)
* |
* Lab

  + [Experiments](https://2021.igem.org/Team%3AUZurich/Experiments)
  + [Engineering Success](https://2021.igem.org/Team%3AUZurich/Engineering)
  + [Notebook](https://2021.igem.org/Team%3AUZurich/Notebook)
  + [Measurement](https://2021.igem.org/Team%3AUZurich/Measurement)
* |
* Human Practices

  + [Integrated Human Practices+ [Implementation](https://2021.igem.org/Team%3AUZurich/Implementation)
    + [Communication](https://2021.igem.org/Team%3AUZurich/Communication)
    + [Education](https://2021.igem.org/Team%3AUZurich/Education)
    + [Sustainability](https://2021.igem.org/Team%3AUZurich/Sustainable)
    + [Entrepreneurship](https://2021.igem.org/Team%3AUZurich/Entrepreneurship)](https://2021.igem.org/Team%3AUZurich/Human_Practices)
* |
* Team

  + [Team](https://2021.igem.org/Team%3AUZurich/Team)
  + [Attributions](https://2021.igem.org/Team%3AUZurich/Attributions)
  + [Contribution](https://2021.igem.org/Team%3AUZurich/Contribution)
  + [Collaboration](https://2021.igem.org/Team%3AUZurich/Collaborations)
  + [Sponsors](https://2021.igem.org/Team%3AUZurich/Sponsors)
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* [Parts](https://2021.igem.org/Team%3AUZurich/Parts)
* |
* [Awards](https://2021.igem.org/Team%3AUZurich/Medal)

# Protocols

## Our Lab Protocols

We used the Benchling notebook software to document our daily progress in the lab and to have protocols for all of our regular
tasks and experiments in one central location. We uploaded the link to the source of the used protocol for a given experiment in the respective file if we have used a protocol from extern.

---

[BSA Protein Quantification Kit](https://static.igem.org/mediawiki/2021/5/51/T--UZurich--BSA_Protein_Quantification_Kit.pdf)

[EtBr room protocol](https://static.igem.org/mediawiki/2021/a/a8/T--UZurich--EtBr_room_protocol.pdf)

[Fusion protocol](https://static.igem.org/mediawiki/2021/7/74/T--UZurich--Fusion_protocol.pdf)

[Lo-12 protocol](https://static.igem.org/mediawiki/2021/c/c5/T--UZurich--Lo-12_protocol.pdf)

[Miniprep protocol](https://static.igem.org/mediawiki/2021/b/b3/T--UZurich--Miniprep_protocol.pdf)

[OMV isolation](https://static.igem.org/mediawiki/2021/4/45/T--UZurich--OMV_isolation.pdf)

[PCR, Digestions and Glycerol stock protocols](https://static.igem.org/mediawiki/2021/9/9c/T--UZurich--PCR_Digestions_and_Glycerol_stock_protocols.pdf)

[ROS protocol](https://static.igem.org/mediawiki/2021/c/c9/T--UZurich--ROS_protocol.pdf)

[SGI protocol](https://static.igem.org/mediawiki/2021/a/a5/T--UZurich--SGI_protocol.pdf)

[Transformation protocol](https://static.igem.org/mediawiki/2021/5/56/T--UZurich--Transformation_protocol.pdf)

![](https://static.igem.org/mediawiki/2021/6/69/T--UZurich--transparent_logo.png)

### UZurich 2021

[Home](https://2021.igem.org/Team%3AUZurich)
[Description](https://2021.igem.org/Team%3AUZurich/Description)
 [Results](https://2021.igem.org/Team%3AUZurich/Results)
[Team](https://2021.igem.org/Team%3AUZurich/Team)
[Human Practices](https://2021.igem.org/Team%3AUZurich/Human_Practices)
[Parts](https://2021.igem.org/Team%3AUZurich/Parts)

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# Team:UZurich/Results

* [iGEM UZurich 2021](https://2021.igem.org/Team%3AUZurich)
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# Results

## **1. Engineered OMVs that trigger stronger immune response**

### Key method

In this part, we mainly performed two plant immunity assays: [seedling growth inhibition (SGI) assay](https://static.igem.org/mediawiki/2021/a/a5/T--UZurich--SGI_protocol.pdf) (Figure 0A, in which lower seedling weight indicates that higher immune responses have been triggered, due to the trade-off between immunity and growth) and [ROS burst assay](https://static.igem.org/mediawiki/2021/c/c9/T--UZurich--ROS_protocol.pdf) (Figure 0B, in which higher ROS production by leaf discs means higher plant immune response) to measure the immune response (Figure 0). CMVs and OMVs were isolated from different bacteria strains, and their immunogenicities are tested on different plant genotypes (see [list of strains and plant genotypes](https://static.igem.org/mediawiki/2021/4/45/T--UZurich--list_strains_genotypes.pdf)).

![](https://static.igem.org/mediawiki/2021/e/e6/T--UZurich--SGI_%26_ROS.png)
*Figure 0: Experimental procedures of seedling growth inhibition and ROS burst assay (modified from [1])*

### **Results**

### ***Natural bacterial membrane vesicles can elicit a strong plant immune response***

First, we wanted to test whether bacteria originated membrane vesicles can trigger plant immune respones. More specifically, which type of vesicle should be used, and what the optimal concentration is. To answer these questions, a seedling growth inhibition assay (SGI, in which lower seedling weight indicates that higher immune responses have been triggered, due to the trade-off between immunity and growth) was performed in *A. thaliana* Col-0 using both outer membrane vesicles (OMVs, natural bulging of outer membrane, thus containing periplasmic contents) and cytoplasmic membrane vesicles (CMVs, generated by explosive cell lysis, thus containing cytoplasmic contents) deriving from the *E. coli* BL21 omp8 strain, an OMV-overproducing strain.

![](https://static.igem.org/mediawiki/2021/8/89/T--UZurich--SGI_omv_cmv_gradient.jpg)
*Figure 1: Plot of SGI experiment data comparing dosage of vesicles*

The performed Two-Way Anova (F(15, 112) = 26.75, p < 2.2e-16, multiple R2 = 0.7818) yielded significance for the concentration (p = 6.23e-10), the vesicle type (p < 2e-16) and the interaction between the two (p = 7.32e-08). This shows that not only the concentration, but also the type of the vesicles have an influence on the strength of the immune response. What's more, the impact of the concentration is dependent on the vesicle type. From a biological viewpoint, ***E. coli* OMVs are more immunogenic than CMVs, therefore OMVs are the better option to use to trigger plant immune responses**, hence we used 5μg/mL OMV to trigger plant immune response for subsequent experiments.

After proving that bacterial membrane vesicles, especially OMVs, can trigger plant immune responses, we came up with questions related to the real-world application:

1. Can we use plant-associated bacteria to produce membrane vesicles in a safer way?
2. Can our vesicles trigger an immune response in different plant species?

We tested the immunogenicity of OMVs and CMVs from three plant-associated bacteria:

1. *A. tumefaciens* GV3101 (disarmed lab strain commonly used for plant transformation)
2. *P. fluorescence* CHA0 (a famous biocontrol strain from Switzerland)[3]
3. *P. putida* IsoF (a plant growth-promoting strain)[4]

Two *A. thaliana* genotypes were used to represent different plants:

1. wildtype Col-0, with an immune receptor called EF-Tu receptor (EFR), representing plants from the Brassicaceae family
2. *efr-1* mutant, without EFR receptor, representing plants that are not from the family Brassicaceae

By conducting a ROS experiment comparing different treatments and their effects on the immune response in different *A. thaliana* genotypes, the Two-way ANOVA (F(13, 154) = 5.558, p = 3.106e-08, multiple R2 = 0.3193) yielded significance for the treatments (p = 1.679e-8) and the interaction (p = 0.01101). This suggests that treatments do have an effect, but there is no difference between the different plant genotypes in their overall immune response to treatments. However, data also suggests that the absence of the EFR receptor affects the immune response, which could imply that the immunogenicity of OMVs from certain strains could be dependent on EF-Tu. If we look at the R2-value, we can see that there are probably explanatory variables missing in the setup/model. **For the real-world application, it means that certain vesicles might not be able to trigger strong immune responses in all crops.**

![](https://static.igem.org/mediawiki/2021/5/5d/T--UZurich--ROS_col0_efr1.jpg)
*Figure 2: Plot of ROS experiment data in A. thaliana Col-0 and efr-1*

Moreover, we wanted to know more about how OMVs trigger the plant immune response, since it can guide us to design OMVs with higher immunogenicity. For this purpose, we tested membrane vesicles with *A. thaliana* lacking different immune receptors. Comparing six different treatments in four different *A. thaliana* genotypes, the Two-way ANOVA (F(23, 160) = 3.903, p = 1.549e-07, multiple R2 = 0.3594) yielded significance for the plants genotype (p = 0.00671), the strain (p = 6.67e-07) and the interaction (p = 0.00347). These results indicate that at least some of the response caused by membrane vesicles is dependent on the presence of certain immune receptors. Post-hoc tests (TukeyHSD) imply that **plant growth-promoting bacteria *P. putida* would be a good choice to produce OMVs**, because **its OMVs induce the strongest immune response among all vesicles tested**, and **its immunogenicity is EFR-independent, implying its potential to trigger a strong immune response in a wide range of plant species**.

![](https://static.igem.org/mediawiki/2021/7/7c/T--UZurich--SGI_4_phenotypes.jpg)
*Figure 3: Plot of SGI experiment data with 4 phenotypes of A. thaliana*

With these preliminary questions answered, let us move on to the data concerning our own engineered vesicles.

### ***Immune response can be further enhanced by displaying certain proteinaceous immune elicitors on the OMV surface***

To verify the functionality of our construct, we performed a ROS assay with a negative control (Water/’Mock’), a functional control (ClyA-only, without any peptide elicitor fused), a positive control (200nM flg22 peptide), the construct ClyA-elf18 ([BBa\_K3989011](http://parts.igem.org/Part%3ABBa_K3989011)) and the construct ClyA-flg22 ([BBa\_K3989010](http://parts.igem.org/Part%3ABBa_K3989010)).

![](https://static.igem.org/mediawiki/2021/6/60/T--UZurich--ROS_by_engineered_OMV.png)
*Figure 4: Plot of ROS experiment data of immune elicitor display constructs (performed post-hoc tests on One-way ANOVA: non-parametric test, Kruskal Wallis test)*

Flg22 ([BBa\_K3286138](http://parts.igem.org/Part%3ABBa_K3286138)) and elf18 ([BBa\_K3989023](http://parts.igem.org/Part%3ABBa_K3989023)) were chosen as the elicitors to be displayed on the OMV surface using ClyA ([BBa\_K811000](http://parts.igem.org/Part%3ABBa_K811000)), because flg22 is recognized as pathogen-associated molecular pattern (PAMP) in many different plants and elf18 is recognized as a potent PAMP in Brassicaceae, which *A. thaliana* belongs to. The Two-way ANOVA showed significant differences between OMVs with and without elicitors. This means that the elicitors on our constructs are crucial for the impact they will have on the plant immune response.

Post-hoc tests (TukeyHSD) have also shown that our constructs can elicit an immune response in *A. thaliana* different from the induced ClyA only, which is a functional negative control that shows if the immune response induced by the constructs is not mediated by ClyA and this suggests that our constructs do in fact work.

To further confirm our results, SGI assays were performed using both *E. coli* BL21 omp8 and *E. coli tolB* strains. Two-way ANOVA results indicate that engineered OMVs are indeed capable of inducing a stronger immune response. The post-hoc (TukeyHSD) indicated that this difference becomes visible from 10µg/ml.

![](https://static.igem.org/mediawiki/2021/f/f6/T--UZurich--SGI_omp8.png)![](https://static.igem.org/mediawiki/2021/d/d4/T--UZurich--SGI_tolB.png)
*Figure 5: Plot of SGI experiment data of immune elicitor display constructs*

### ***OMVs with cellulose-binding domain can also trigger stronger immune responses***

In addition to the surface display of plant immune elicitors, we also sought for other strategies to improve the performance of our OMVs for agricultural application. During our integrated human practices, a farmer told us that pesticides applied to plants are often washed away by rain. To solve this problem, we came up with the idea to make OMVs better attach to plant tissue by displaying a cellulose-binding domain on the surface of the OMVs. Although we had no time to show that engineered OMVs with dCBD are washed off by rain less extensively, we found that they can trigger stronger immune responses. As shown in Figure 6, the seedling weight of the negative control group gradually decreases as OMV concentration increases. However, for the dCBD group, the seedling weight dramatically decreases between concentrations of 0 and 2.5 but doesn't decrease a lot as the concentration further increases, which means the immune response might get saturated.

One possible explanation for such results is the enrichment of OMVs by cellulose in the cell wall. At low concentrations, the cell wall limits the diffusion of wild-type OMVs, but for engineered OMVs with dCBD, they get enriched in the cell periphery, generating a higher local concentration and facilitating the diffusion. At higher concentrations, the enrichment effect gets saturated, and OMVs attached to cellulose can even block free OMVs from entering, resulting in no difference between ClyA-dCBD and ClyA-only groups.

![](https://static.igem.org/mediawiki/2021/0/03/T--UZurich--SGI_ClyA-dCBD.jpg)
*Figure 6: Plot of SGI experiment data of cellulose binding domain display constructs*

### **Conclusions**

1. To induce plant immune responses, OMVs are a better choice than CMVs, due to their high immunogenicity.
2. Plant growth-promoting bacteria *P. putida* is a good chassis for OMV production in a real-world application, since it can probably induce a high immune response in most crops.
3. Our engineered OMVs with either immune elicitor or cellulose-binding domain displayed on surface can trigger stronger plant immune responses than natural OMVs.

## **2. CMV-based delivery system**

Additionally to the main part of our project, we also conducted the fusion experiment. For this experiment we were advised and supervised by Professor Olivier Voinnet, an RNA silencing expert at the ETH.

We wanted to find out if vesicles could fuse with and therefore transfer DNA into plant cells. We used CMVs rather than OMVs since they contain all cell contents, whereas OMVs only contain periplasm.
Professor Voinnet suggested using one of his viral vectors containing only GFP and a replicase, which will amplify the GFP signal in a single cell by replicating itself. In addition, we used P19, an RNAi blocker, to inhibit the plant's antiviral response. We infiltrated wild type *N. benthamiana* with 4 different treatments:

1. positive control, 1:1 mix of agrobacterium with the viral vector and agrobacterium with P19. 2 leaves were infiltrated.
2. 1:1 mix of E.coli (BL21 DH10b) with the viral vector and with P19, 2 leaves were infiltrated.
3. 1:1 mix of CMVs from (BL21 DH10b) with the viral vector and CMVs with P19, the CMVs were prepared using MMC as a mutagen to induce cell lysis. 1 leaf was infiltrated.
4. We used plain Buffer (MES/MgCl2) as a negative control. We are aware that this is not the most suitable negative control, but we did not have E.coli with an unrelated plasmid prepared. 2 leaves were infiltrated.

We used new gloves and syringes for each infiltration to minimise risk of contamination during infiltration.
Since this viral vector is non-virulent, meaning it can’t spread systemically, we knew even if a fusion happens it would only be the case in isolated cells. Therefore we looked at our plants after 1 week under the confocal microscope (as soon as the positive control was visibly green under UV light).

![](https://static.igem.org/mediawiki/2021/7/78/T--UZurich--fusion_fig1.png)

*Figure 7: Positive control from the fusion experiment*

This is the positive control, you see the cells are packed with GFP, which makes sense, since agrobacterium is an
established DNA delivery system.

![](https://static.igem.org/mediawiki/2021/8/80/T--UZurich--fusion_fig2.png)

*Figure 8: E.coli infiltrated cell from the fusion experiment*

This is an epidermal cell from an *E. coli* infiltrated leaf. The GFP illuminates the nucleus and the cytosol, which are pressed to the cell borders by the vacuole.
We did not do exact cells per area counts, but we took 4-5 samples of about a 1x2 cm size and saw 5-10 fluorescent single cells.

![](https://static.igem.org/mediawiki/2021/0/0f/T--UZurich--fusion_fig3.png)

*Figure 9: CMV infiltrated leaf from the fusion experiment*

This is from a CMV infiltrated leaf, where we did not find evidence for a fusion in any of the CMV samples. This however does not necessarily mean that a fusion is impossible, it could also be due to our mutagen damaging the plasmids.

In all of the samples there was some autofluorescence, as well as fluorescence around the site of infiltration. However, these were clearly distinguishable from actual fluorescent live cells.

### **Conclusion of the first experiment**

Could *E. coli* possibly possess a delivery system, allowing the transfer of DNA between plant and *E. coli* cells? Due to the small sample size and lack of an appropriate negative control, we can not conclude anything. Our result could also stem from contamination. Therefore, we repeated the experiment to see whether we could replicate our results. However, we used a different mutagen (UV rays) to prepare our CMVs, since the chemical mutagen (MMC) stays in the plant and keeps on mutagenising, which could damage our construct. For more information on the general process see our [protocol](https://static.igem.org/mediawiki/2021/7/74/T--UZurich--Fusion_protocol.pdf) for the first fusion experiment.

### **Results of the repeated experiment**

![](https://static.igem.org/mediawiki/2021/a/ad/T--UZurich--fusion_fig4.jpg)

*Figure 10: Positive control (A. tumefaciens)*

In this figure we can see clear fluorescence as in the positive control in the first experiment. Thus our construct seems to work.

![](https://static.igem.org/mediawiki/2021/5/54/T--UZurich--fusion_fig5.jpg)

*Figure 11: E. coli BL21*

In this figure we can only see some autofluorescence of the plant organelles. The infiltrate was the *E. coli* strain BL21 which is a common lab strain, which we also used during our project. We cannot see any evidence for DNA delivery in this experiment.

![](https://static.igem.org/mediawiki/2021/4/43/T--UZurich--fusion_fig6.jpg)

*Figure 12: E. coli DH10b*

This infiltrate consists of the same *E. coli* strain (*DH10b*), that seemed to induce fluorescence in the first experiment, however in this repeat, we could not find any evidence for DNA delivery into the plant through this bacterium.

![](https://static.igem.org/mediawiki/2021/8/8a/T--UZurich--fusion_fig7.jpg)

*Figure 13: CMVs*

As in the first experiment, we could not find any evidence for DNA delivery into the plant, through our CMVs.

![](https://static.igem.org/mediawiki/2021/b/bd/T--UZurich--fusion_fig8.jpg)

*Figure 14: CMVs without p19*

We also tested the CMVs without an P19 background. Here as well, one can only see a small amount of autofluorescence but no evidence for DNA delivery into the plant.

![](https://static.igem.org/mediawiki/2021/2/22/T--UZurich--fusion_fig9.jpg)

*Figure 15: Negative control 1 (E. coli lysate)*

We included the cell lysate of *E. coli* as a negative control to see whether in our first experiment, the DNA was released as the bacteria lysed inside the plant’s interstitium and could be taken up even without any living bacteria. Here as well, we couldn’t detect any DNA uptake by the plant epidermal cells.

![](https://static.igem.org/mediawiki/2021/7/79/T--UZurich--fusion_fig10.jpg)

*Figure 16: Negative control 2 (Pure viral vector DNA)*

To also test whether the plant epidermal cells could just take up pure viral vector DNA, we also tested a second negative control which was an infiltration, with just the vector. Here we can also see no DNA uptake by the plant.

### **Conclusion of repeated experiment**

After analysing our samples from the second experiment, we could not find any evidence for induced fluorescence in any of our samples, except for the positive control. Thus we conclude that there was probably some kind of contamination in the first experiment that led to the fluorescence. With the experiment repeated, we could not find any evidence for any DNA delivery into the plant epidermal cells through either CMVs, OMVs, *E. coli BL21*, *E. coli DH10b*, pure viral vector or *E. coli* lysate with viral vector.

## **3. Inducible OMV production & Improvement of an existing part**

### ***Characterization of a Quorum Sensing regulator protein EsaR and its variants***

The core of our inducible OMV production system (see our
[Design](https://2021.igem.org/Team%3AUZurich/Design) page) is the repression of TolB expression when engineered bacteria perceive quorum sensing molecules from pathogens. This process is mediated by the EsaR regulator and PesaS promoter.

Previous teams have already introduced the wild type EsaR protein ([BBa\_K2116001](http://parts.igem.org/Part%3ABBa_K2116001)) and its basic information. In our project, we characterized its function of regulating the downstream gene transcription based on the amount of AHL molecule added. Together with the wild-type, we also characterized three variants of this protein: EsaR-I70V ([BBa\_K3989003](http://parts.igem.org/Part%3ABBa_K3989003)), EsaR-D91G ([BBa\_K3989004](http://parts.igem.org/Part%3ABBa_K3989004)) and EsaR-V220A ([BBa\_K3989005](http://parts.igem.org/Part%3ABBa_K3989005)).

The construct we used to characterize these parts with is a combination of the EsaR protein, promoter of the protein PesaS ([BBa\_3989009](http://parts.igem.org/Part%3ABBa_K3989009)) and a GFP protein ([BBa\_3989025](http://parts.igem.org/Part%3ABBa_K3989025)). The promoter we used, once activated, starts the transcription when EsaR binds to it. But when there are AHL molecules in the environment, EsaR will interact with them instead and dislocate from the promoter. Thus, the transcription will be suppressed or even blocked when the concentration of the AHL molecules is high.

From our characterization, we found that the D91G and V220A variants are more sensitive to the AHL molecules and I70V shows a similar sensitivity compared to wild-type.

![](https://static.igem.org/mediawiki/2021/e/e5/T--UZurich--improvement_1.jpeg)

*Figure 17: Fluorescence intensity measured by plate reader (after 7 hours' induction).*

![](https://static.igem.org/mediawiki/2021/7/7e/T--UZurich--improvement_2.jpeg)
*Figure 18: Fluorescence intensity measured by flow cytometry. The samples are taken after 7 hours' induction.*

### [**go to top**](#results-image)

---

[1] Bredow, M., Sementchoukova, I., Siegel, K., & Monaghan, J. (2019). Pattern-triggered oxidative burst and seedling growth inhibition assays in Arabidopsis thaliana. JoVE (Journal of Visualized Experiments), (147), e59437.

[2] Bahar, Ofir, et al. "Bacterial outer membrane vesicles induce plant immune responses." Molecular Plant-Microbe Interactions 29.5 (2016): 374-384.

[3] Keel, C., Schnider, U., Maurhofer, M., Voisard, C., Laville, J., Burger, U., ... & Défago, G. (1992). Suppression of root diseases by Pseudomonas fluorescens CHA0: importance of the bacterial secondary metabolite 2, 4-diacetylphloroglucinol. Molecular Plant-Microbe Interactions, 5(1), 4-13.

[4] Steidle, A., Allesen-Holm, M., Riedel, K., Berg, G., Givskov, M., Molin, S., & Eberl, L. (2002). Identification and characterization of an N-acylhomoserine lactone-dependent quorum-sensing system in Pseudomonas putida strain IsoF. Applied and Environmental Microbiology, 68(12), 6371-6382.

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### UZurich 2021

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