iGEM 2018 — Team BOKU-Vienna

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

# Team:BOKU-Vienna/Notebook

## Notebook

# June: Preparation for Wet lab phase

6.06 Sowing of Arabidopsis thaliana

18.06-29.06 Design of Golden Gate compatible

# July

## Week 1 2.07-6.07

Lab Safety instructions from our Supervisors

Ordering chemicals, further primers and required plasmids

Production of media, buffers and agarose gels

Production of chemically competent E.coli

We have received transformed E.coli with the Golden Gate backbones from our PI.

Plasmid preparation of Golden Gate backbones 1 (BB1 FS1-FS2, BB1 FS2-FS3, BB1 FS3-FS4) and 2 (BB2 FSA-FSB, BB2 FSB-FSC, BB2 FSC-FSD, BB2 FSD-FSE)

Unfortunately, restriction analysis did not produce a good result because alkaline lysis did not work as intended. The samples have a very low concentration and are heavily contaminated.

[![Plasmid preparation of Golden Gate backbones 1 <sub>(BB1 FS1-FS2, BB1 FS2-FS3, BB1 FS3-FS4)</sub> and 2 <sub>(BB2 FSA-FSB, BB2 FSB-FSC, BB2 FSC-FSD, BB2 FSD-FSE)</sub>](https://static.igem.org/mediawiki/2018/b/b1/T--BOKU-Vienna--2018_pic1.png)](https://2018.igem.org/Team%3ABOKU-Vienna/pic1.png)

## Week 1 2.07-6.07

That week was packed with work. Doris and Theresa started InterLab study and had some difficulties, because they took a 50µl instead of 500µL sample at timepoint zero. Two days later they were able to repeat the experiment and succeeded.

#### Renewed plasmid preparation of Golden Gate backbones 1 (BB1 FS1-FS2, BB1 FS2-FS3, BB1 FS3-FS4) and 2 (BB2 FSA-FSB, BB2 FSB-FSC, BB2 FSC-FSD, BB2 FSD-FSE)

[![Renewed plasmid preparation of Golden Gate backbones 1 <sub>(BB1 FS1-FS2, BB1 FS2-FS3, BB1 FS3-FS4)</sub> and 2 <sub>(BB2 FSA-FSB, BB2 FSB-FSC, BB2 FSC-FSD, BB2 FSD-FSE)</sub>](https://static.igem.org/mediawiki/2018/0/02/T--BOKU-Vienna--2018_pic2.png)](https://2018.igem.org/Team%3ABOKU-Vienna/pic2.png)

1. Ladder
2. Empty
3. BB1 FS1-FS2
4. BB1FS2-FS3
5. BB1FS3-FS4
6. Empty
7. BB2 FSA-FSB
8. BB2 FSB-FSC
9. BB2 FSC-FSD
10. BB2 FSD-FSE

#### PCR of MRE – elements Anneal Tm = 72 °C

[![PCR of MRE – elements <sub>Anneal Tm = 72 °C</sub>](https://static.igem.org/mediawiki/2018/9/90/T--BOKU-Vienna--2018_pic3.png)](https://2018.igem.org/Team%3ABOKU-Vienna/pic3.png)

1. Ladder
2. MRE1
3. MRE2
4. MRE3
5. MRE4

#### PCR of AlcA (Anneal Tm = 67 °C)

PCR of AlcR mut1bw (Anneal Tm = 70 °C) und AlcR mut1fw (Anneal Tm = 68 °C)

PCR of Cup – elements: CUP2 mut1bw & CUP2 mut2bw (Anneal Tm = 67 °C), CUP2 mut2fw (Anneal Tm = 65 °C)

The PCR of Cup and AlcA did not work according to gel electrophoresis, therefore AlcA was tested in several different ways (With Enhancer and bwPrimer, without Enhancer and bW Primer, with Enhancer and bw 35 Primer, without Enhancer and bw 35 Primer).

However, these unfortunately didn’t work either.

## Week 3 16.07-20.07

We received our ordered plasmids from iGEM and immediately continued working with them.

PCR of p35S -830 (Anneal Tm = 66 °C), p35S -90 (Anneal Tm = 63 °C)

PCR of uGFP: uGFP gRNA\_mut1fw & uGFP mut1fw (Anneal Tm = 68 °C), uGFP mut1\_ mut2 (Anneal Tm = 68 °C), uGFP mut2bw (Anneal Tm = 67 °C)

PCR of VP16 (Anneal Tm = 70 °C)

PCR of cup elements: Repeat week 2 experiment

[![PCR of cup elements: Repeat week 2 experiment](https://static.igem.org/mediawiki/2018/1/1a/T--BOKU-Vienna--2018_pic4.png)](https://2018.igem.org/Team%3ABOKU-Vienna/pic4.png)

1. Ladder
2. Cup1 (not ok)
3. Cup2
4. Cup3
5. GFP gRNA 1
6. GFP 2
7. GFP 3
8. GFP 4
9. Ladder

This week we did our first Golden Gate cloning:

BB1\_FS1\_p35S-830\_MREx4\_p35S-90\_FS2

BB1\_FS2\_AlcR mut1fw\_ AlcR mut1bw\_FS3

BB1\_FS2\_uGFP\_FS3

According to restriction analysis the AlcR transformation was a success, but the MRE and GFP transformation has not.
On Friday Johannes, Doris, Michaela und Philipp traveled to the European meetup in Munich. They had a lot of fun and could exchange their experiences they had so far with other teams.

## Week 4 23.07-27.07

This week we start with the first PCR for our toggle switch and try hard to get the other things going, which haven’t worked yet.

PCR of MRE (Anneal Tm = 72 °C)

PCR of p35S -830 (Anneal Tm = 66 °C), p35S -90 (Anneal Tm = 63 °C), p35S -46 Anneal Tm = 57 °C

PCR of uGFP: uGFP gRNA\_mut1fw uGFP mut1fw (Anneal Tm = 68 °C), uGFP mut1\_ mut2 (Anneal Tm = 68 °C), uGFP mut2bw (Anneal Tm = 67 °C)

PCR of gTargetOff: gTargetOff1, gTargetOff2, gTargetOff3, gTargetOff4 (Anneal Tm = 70 °C)

PCR of gTargetOff: gTargetON1, gTargetON2, gTargetON3(Anneal Tm = 67 °C), gTargetON4 (Anneal Tm = 64 °C)

PCR of LexA (Anneal Tm = 58 °C)

#### Overlapping Extension PCR of gRNA ON, gRNA Off (Anneal Tm = 72 °C)

[![PCR of gRNA Element and gTarget Element](https://static.igem.org/mediawiki/2018/8/84/T--BOKU-Vienna--2018_pic5.png)](https://2018.igem.org/Team%3ABOKU-Vienna/pic5.png)

1. Ladder
2. gRNA ON
3. gRNA OFF
4. Empty
5. gTarget ON
6. gTarget ON
7. gTarget ON
8. Empty
9. p35S -830
10. Ladder

[![PCR of p35S Element](https://static.igem.org/mediawiki/2018/3/3a/T--BOKU-Vienna--2018_pic6.png)](https://2018.igem.org/Team%3ABOKU-Vienna/pic6.png)

1. p35S -90
2. Ladder

[![PCR of gTargetOFF Elements](https://static.igem.org/mediawiki/2018/5/5a/T--BOKU-Vienna--2018_pic7.png)](https://2018.igem.org/Team%3ABOKU-Vienna/pic7.png)

1. Ladder
2. gTarget OFF1
3. gTarget OFF2
4. gTarget OFF3
5. gTarget OFF4

We have received several terminators and promoters (BB1\_12\_PRPP16, BB1\_34\_RPS25, BB1\_34\_RP25TT, BB1\_34\_RPL2, BB1\_12\_RPT2F, BB1\_12\_PGAP, BB1\_12\_PRPP16, BB1\_34\_RPS25, BB1\_34\_RPL2, BB1\_34\_RP25TT)
and Golden Gate backbones 3(BB3 FSA-FSC, BB3 FSA-FSD) from our supervisor.

We also had synthetic components made for the XVE element, which we got on Monday. So we were able to clone them in BB1 right away.

#### Golden Gate cloning:

* BB1\_FS1\_p35S-46\_LexA\_FS2
* BB1\_FS2\_XVE1-5\_FS3
* BB1\_FS2\_gTargetOff\_uGFP\_FS3
* BB1\_FS2\_ uGFP\_FS3
* BB1\_FS2\_Cup\_VP16\_FS3
* BB1\_FS1\_p35S-830\_MREx4\_p35S-90\_FS2

According to restriction Analysis the vectors showed no right insert.

# August

## Week 6 30.07-3.08

Since almost nothing has worked so far, we had a lot of troubleshooting and we came to the conclusion, that we may interchanged the BB1 when purifying it. That's why we used a BB1 from last year's iGEM team for comparison in Golden Gate cloning.
Additionally, we started to amplify promoters and terminators for plants (MAS, tNOS, p35S)

* PCR of MAS (Anneal Tm = 68 °C)
* PCR of tNOS (Anneal Tm = 71 °C)
* PCR of p35S (Anneal Tm = 62 °C)

1. Ladder
2. p35S
3. MAS
4. tNOS
5. Ladder

[![PCR of p35S, MAS and tNOS](https://static.igem.org/mediawiki/2018/7/70/T--BOKU-Vienna--2018_pic8.png)](https://2018.igem.org/Team%3ABOKU-Vienna/pic8.png)

#### Production of competent P.pastoris

#### Golden Gate cloning:

We made the uGFP Golden Gate experiment with the Backbone we purified our own and the same Backbone from summer 2017.

* BB1\_FS2\_gTargetOff\_uGFP\_FS3
* BB1\_FS2\_uGFP\_FS3- 2018 / BB1\_FS2\_uGFP\_FS3- 2017
* BB1\_FS1\_MAS\_FS2
* BB1\_FS1\_p35S\_FS2
* BB1\_FS3\_tNOS\_FS4

1. Ladder
2. BB1 uGFP-2017-1

[![Restriction analysis of uGFP in BB1 from iGEM-Team 2017](https://static.igem.org/mediawiki/2018/e/ef/T--BOKU-Vienna--2018_pic9.png)](https://2018.igem.org/Team%3ABOKU-Vienna/pic9.png)

## Week 7 6.08-10.08

This week was a big success. We sent in several parts for sequencing and got five positive results.

#### Golden Gate cloning:

We used new purified BB1

* BB1\_FS2\_XVE1-5\_FS3
* BB1\_FS3\_tNOS\_FS4
* BB1\_FS1\_p35S-46\_LexA\_FS2
* BB1\_FS1\_p35S-830\_MREx4\_p35S-90\_FS2
* BB1\_FS1\_p35S-46\_AlcA\_FS2
* BB1\_FS2\_gTargetON-gRNAOFF\_FS3
* BB1\_FS2\_gTargetOFF\_gRNAON\_FS3
* BB1\_FS2\_dCas9\_FS3
* BB1\_FS2\_Cup\_VP16\_FS3
* pPLV03 FSA-FSB
* pPLV03 FSA-FSC
* pPLV03 FSA-FSD
* BB2\_uGFP
* BB3\_uGFP

We got positive sequencing Results for BB1\_FS1\_p35S-46\_LexA\_FS2, BB1\_FS1\_p35S-46\_AlcA\_FS2, BB1\_FS2\_ AlcR mut1fw\_ AlcR mut1bw \_FS3, BB1\_FS2\_dCas9\_FS3, BB1\_FS2\_Cup\_VP16\_FS3
The MRE construct has lost one element during cloning.

## Week 8 13.08-17.08

On Monday we held a presentation at a company called BIOMIN, which later became one of our sponsors.
Two days later Ulrich visited an apple farmer to discuss our project with him.

PCR of UBQ10 promoter: pUBQ10 FS1 (Anneal Tm = 63 °C), pUBQ10 FS2 (Anneal Tm = 64 °C)

PCR of UBQ10 terminator (Anneal Tm = 64 °C)

PCR of WUS terminator (Anneal Tm = 61 °C)

PCR of RBCS terminator (Anneal Tm = 65 °C)

PCR did not work well. We tried it again the following week.

[![PCR: UBQ10p, UBQ10t, WUSt, RBCSt](https://static.igem.org/mediawiki/2018/d/df/T--BOKU-Vienna--2018_pic10.png)](https://2018.igem.org/Team%3ABOKU-Vienna/pic10.png)

1. Ladder
2. UBQ10 FS1
3. UBQ10 FS2
4. tUBQ10
5. tWUS
6. tRBCS
7. Ladder

#### Golden Gate Cloning:

BB1\_FS2\_XVE1-5\_FS3

BB1\_FS2\_gTargetON-gRNAOFF\_FS3

BB1\_FS2\_gTargetOFF\_gRNAON\_FS3

BB1\_FS2\_gTargetOff\_uGFP\_FS3

BB1\_FS1\_p35S-830\_MREx4\_p35S-90\_FS2

According to restriction Analysis the vectors showed not the desired insert.

#### Transformation of BB3\_uGFP in P.pastoris

We got positive sequencing Results for BB1\_FS3\_tNOS\_FS4 and pPLV03 FSA-FSB

[![Restriction Result BB1_FS3_tNOS_FS4](https://static.igem.org/mediawiki/2018/b/b7/T--BOKU-Vienna--2018_pic11.png)](https://2018.igem.org/Team%3ABOKU-Vienna/pic11.png)

1. Ladder
2. BB1 tNOS - no insert
3. BB1 tNOS - right insert

## Week 9 20.08- 24.08

Due to the fact that the parts MRE, XVE, gTargetOFF-uGFP, gTargetOFF-gRNAON, gTargetON-gRNAOFF, still didn’t work and the sequencing results show that they lost single individual parts, we had another trouble shooting with our PI.

All these parts consist of four or more individual parts. So, we try to fuse 2-3 individual parts before cloning them altogether into the vector.

BB1\_FS2\_XVE1-5\_FS3

BB1\_FS2\_gTargetON-gRNAOFF\_FS3

BB1\_FS2\_gTargetOFF\_gRNAON\_FS3

BB1\_FS1\_p35S-830\_MREx4\_p35S-90\_FS2

BB1\_FS2\_gTargetOff\_uGFP\_FS3

In addition to this we tried to produce guideRNA for our liposome experiment, but we got no bands on the Gel.

#### PCR of UBQ10 promoter:

pUBQ10 FS1 (Anneal Tm = 63 °C), pUBQ10 FS2 (Anneal Tm = 64 °C)

PCR of UBQ10 terminator (Anneal Tm = 64 °C)

PCR of WUS terminator (Anneal Tm = 61 °C)

PCR of RBCS terminator (Anneal Tm = 65 °C)

PCR of p35S-830 (Anneal Tm = 66 °C)

PCR of MAS (Anneal Tm = 68 °C)

[![PCR](https://static.igem.org/mediawiki/2018/e/e1/T--BOKU-Vienna--2018_pic12.png)](https://2018.igem.org/Team%3ABOKU-Vienna/pic12.png)

1. p35S-830
2. Ladder

[![PCR](https://static.igem.org/mediawiki/2018/0/08/T--BOKU-Vienna--2018_pic13.png)](https://2018.igem.org/Team%3ABOKU-Vienna/pic13.png)

1. Ladder
2. pMAS

[![PCR](https://static.igem.org/mediawiki/2018/1/17/T--BOKU-Vienna--2018_pic14.png)](https://2018.igem.org/Team%3ABOKU-Vienna/pic14.png)

1. Ladder
2. pUBQ10 FS1
3. pUBQ10 FS2
4. tRBCS

[![PCR](https://static.igem.org/mediawiki/2018/d/d7/T--BOKU-Vienna--2018_pic15.png)](https://2018.igem.org/Team%3ABOKU-Vienna/pic15.png)

1. Ladder
2. tUBQ10

[![PCR](https://static.igem.org/mediawiki/2018/d/d2/T--BOKU-Vienna--2018_pic16.png)](https://2018.igem.org/Team%3ABOKU-Vienna/pic16.png)

1. Ladder
2. BB1p35S - NciI
3. BB1p35S - Bsp1286i

#### Golden Gate cloning

BB1\_FS1\_MAS\_FS2

BB1\_FS3\_RBSC\_FS4

BB1\_FS2\_tUBQ10\_FS3

BB1\_FS1\_pUBQ10\_FS2

BB1\_FS1\_p35S\_FS2

BB2\_FSB\_AlcA\_GFP\_rpl2\_FSC

BB2\_FSA\_GAP\_AlcR\_RPS25\_FSB

BB2\_FSB\_LexA\_GFP\_rpl2\_FSC

BB2\_FSA\_pGAP\_CUP2\_VP16\_RPS25\_FSB

We did a colony PCR on BB1\_FS2\_XVE1-5\_FS3, BB1\_FS2\_gTargetON-gRNAOFF\_FS3, BB1\_FS2\_gTargetOFF\_gRNAON\_FS3, BB1\_FS1\_p35S-830\_MREx4\_p35S-90\_FS2, BB1\_FS2\_gTargetOff\_uGFP\_FS3 but had no positive result again.

We got positive sequencing Results for B1\_FS1\_p35S\_FS2.

## Week 10 27.08-31.08

Ulrich and Johannes had a meeting with Timo Küntzle, a journalist and organic farmer, to discuss our project with him.

A colleague of our PI, Dominik, showed Ulrich how to make liposomes and Theresa tried to produce guideRNA a second time.

Benedikt ordered new synthetic components for our XVE part, and made a new Golden Gate protocol for it with a three times higher insert concentration.

#### Golden Gate cloning:

BB2\_FSA\_p35S\_dCas9\_tNOS\_FSB

BB3\_FSA\_GAP\_AlcR\_RPS25\_ AlcA\_GFP\_rpl2\_FSB

pPLV03\_A\_p35S\_uGFP\_tNOS\_B

We got positive sequencing Results for B1\_FS2\_tUBQ10\_FS3.

# September

## Week 11 3.09-7.09

We are still trying to fix our problems with the Toggle-Switch parts and the MRE part.

The iGEM Team Marburg sent us their Vibrigens InterLab Meassurement kit.

The sequencing of the WUS terminator showed us that it has several mutations, so we had to strike it of our list.

On Friday Johanna and Theresa transformed pPLV03\_A\_p35S\_uGFP\_tNOS\_B in Agrobacterium tumefaciens. Now, nothing was standing in our way anymore, to transform the first plants next week.

Transformation of BB3\_FSA\_GAP\_AlcR\_RPS25\_ AlcA\_GFP\_rpl2 \_FSB into P.pastoris

The new Golden Gate protocol for the XVE elements worked, and we finally got positive sequencing results for BB1\_FS2\_XVE1-5\_FS3 and also for B1\_FS1\_pUBQ10\_FS2.

[![Restriction Analysis](https://static.igem.org/mediawiki/2018/3/37/T--BOKU-Vienna--2018_pic17.png)](https://2018.igem.org/Team%3ABOKU-Vienna/pic17.png)

## Week 12 10.09-14.09

Doris and Michaela start the measurement for the Vibrigens InterLab.

Philipp, David and Theresa transformed pPLV03\_A\_p35S\_uGFP\_tNOS\_B into Arabidopsis thaliana.

This week we started with the golden gate cloning for the remaining plant parts.

We also designed new primers, developed a new protocol for the Toggle Switch parts (gTargetON-gRNAOFF, gTargetOFF-gRNAON, gTargetOFF-uGFP) and ordered synthetic components as a template for our guide RNAs.

Additional we begun the testing of the ethanol receptor system (BB3\_FSA\_ GAP\_AlcR\_RPS25\_ AlcA\_GFP\_rpl2 \_FSB) in P.pastoris, but did not get any mentionable results.

#### Overlapping extension PCR:

gTarget ON-PCR-1 und gTarget ON-PCR-2 (Anneal Tm = 61°)

gTarget ON-PCR-3 und gTarget ON-PCR-4 (Anneal Tm = 64 °C)

gTarget OFF-PCR-1 und gTarget OFF-PCR-2 (Anneal Tm = 66 °C)

gTarget OFF-PCR-3 und gTarget OFF-PCR-4 (Anneal Tm = 65 °C)

gRNA ON (Anneal Tm = 72 °C)

gRNA OFF (Anneal Tm = 65 °C)

#### Golden Gate cloning

:

BB2\_FSB\_AlcA\_GFP\_RBCS\_FSC

BB2\_FSA\_p35S\_AlcR\_tNOS\_FSB

BB2\_FSB\_LexA-GFP-RBCS\_FSC

BB2\_FSA\_p35S\_CUP\_tNOS\_FSB

BB2\_FSA\_GAP\_CUP\_tNOS\_FSB

## Week 13 17.09-21.09

#### Another successful week.

Our Toggle Switch parts (gTargetON-gRNAOFF, gTargetOFF-gRNAON, gTargetOFF-uGFP) were positively sequenced and the production of the guideRNA with the new templates worked well. The red arrows point out the positive colonies, which were sent in for sequencing.

[![Successfull Week](https://static.igem.org/mediawiki/2018/b/b0/T--BOKU-Vienna--2018_pic18.png)](https://2018.igem.org/Team%3ABOKU-Vienna/pic18.png)

Philipp and Ulrich, produced new Liposomes with a different lipid content and which contained guideRNAON or guideRNAOFF.

Michaela has transformed all parts that have already been sequenced into E.coli and made masterplates and cryostock cultures from them.

This week was the 10. Birthday of the ÖGMBT. In the course of which we were allowed to exhibit our poster. Philipp and Shadan took care of our stand and told us that we got a lot of encouragement of different scientists.

#### Golden Gate cloning:

BB2\_FSB\_TEF\_gTargetON-gRNAOFF\_rpl2\_FSC

BB2\_FSC\_pUBQ10\_gTargetON\_gRNAOFF\_tUBQ10\_FSD

BB2\_FSA\_GAP\_gTOFF\_uGFP\_rps25\_FSB

BB2\_FSB\_p35S\_gTOFF\_uGFP\_rbcs\_FSC

BB2\_FSC\_rpp\_gTOFF\_gRNAON\_rps25\_FSD

BB2\_FSD\_p35S\_gTOFF\_gRNAON\_tUBQ10\_FSC

BB2\_FSB\_LexA\_gRNAON/gRNAOFF\_rpl2\_FSC

BB2\_FSB\_AlcA\_gRNAON/gRNAOFF\_rpl2\_FSC

pPLVO3\_A-E

pPLV03\_FSA\_GAP\_AlcR\_rpl25\_AlcA\_GFP\_rpl2\_FSB

We started our liposome fusion experiment, in which we tested if P.pastoris absorbs liposomes enclosed fluorophore. A colleague of our PI, Thomas, showed Benedikt how to work with the fluorescent microscope.

The iGEM Team Stockholm sent us their parts and we designed and ordered primers for their parts.

## Week 13 24.09-28.09

We designed suitable primers for our inserts to ligate them into the pSB1C3 vector. Our inserts had some forbidden restriction sites, so we had to mutate them via PCR.

We had the idea to mutate the vector in a way, that we could use it in a Golden Gate assembling.

Finally, our MRE elements worked and we got positive sequencing results for BB1\_FS1\_p35S-830\_MREx4\_p35S-90\_FS2. We solved this problem by pre-ligating the individual MRE elements and then cloning them into the backbone.

Doris und Theresa repeated the measuring of the ethanol system in *P.pastoris*, but some of the shake flasks broke on the shaker, so they were not able to finish their experiment.

For the first time, David was able to catch some good photos on the fluorescent microscope of the Liposome fusion experiment.

[![Successfull Week](https://static.igem.org/mediawiki/2018/b/b2/T--BOKU-Vienna--2018_pic19.jpg)](https://2018.igem.org/Team%3ABOKU-Vienna/pic19.jpg)

PCR for Laccase (Anneal Tm = 72°)/ (Anneal Tm = 62°)

PCR for mutated Laccase (Anneal Tm = 72°)/ (Anneal Tm = 72°)

Ulrich and Philipp produced liposomes containing fluorophore.

On Friday, we had to move to another laboratory, since the one where were in summer is needed again, so we were very busy tidying up, clearing out and cleaning up.

In that week we were also generating the fragments for our Sweden-collaboration. For this we ran a PCR to obtain Laccase and Mutated Laccase via gel electrophoresis. We also obtained an alpha Leader sequence

[![Laccase PCR](https://static.igem.org/mediawiki/2018/9/95/T--BOKU-Vienna--2018_pic20.png)](https://2018.igem.org/Team%3ABOKU-Vienna/pic20.png)

1. Laccase
2. Laccase
3. Laccase
4. Laccase
5. Leader
6. Ladder
7. Mut. Laccase
8. Mut. Laccase
9. Mut. Laccase
10. Mut. Laccase

This was followed by a Golden Gate cloning to get the parts into the BB1 backbone. According to a restriction analysis the transformation did not work. The cause was wrong primer design so we ordered new primers.

[![BB1 Laccase](https://static.igem.org/mediawiki/2018/5/59/T--BOKU-Vienna--2018_pic21.jpg)](https://2018.igem.org/Team%3ABOKU-Vienna/pic21.jpg)

1. Ladder
2. Mut. Laccase
3. Mut. Laccase
4. Mut. Laccase
5. Mut. Laccase
6. Laccase
7. Laccase
8. Laccase
9. Laccase
10. Mut. Laccase
11. Mut. Laccase
12. Laccase
13. Laccase
14. Ladder

# October

## Week 14 1.10-5.10

We had big problems with the PCRs for the iGEM parts, and still could not mutate the vector pSB1C3 to use it in Golden Gate cloning.

Theresa tagged the guideRNA with fluorophore, so that we could measure if P.pastoris absorbs the liposomes containing guideRNA.

David and Doris mixed empty liposomes with gRNAON or gRNAOFF and in each case also with the tagged version.

#### Golden Gate cloning

BB2\_FSB\_MRE\_GFP\_rpl2\_FSC

BB3\_FSA\_gTargetOFF\_uGFP\_gTargetON\_gRNAOFF\_FSB

BB3\_FSA\_gTargetOFF\_uGFP\_FSC

BB3\_FSA\_GAP\_XVE\_rps25\_LexAGFP\_rpl2\_FSC

Michaela transformed *P.pastoris* with the whole Toggle switch.

(BB3\_ FSA\_gTargetOFF\_uGFP\_gTargetON\_gRNAOFF\_gTargetOFF\_gRNAON\_FSD)

## Week 15 8.10-10.10

On monday we changed our strategy so that we can submit at least two parts to the iGEM vector in time for the submission deadline. We digested the vector and the inserts with EcoRI and PstI. Then we ligated them with T4 ligase and transformed them into E.coli so we could do the plasmid preparation on Wednesday to send them in instead of doing it by Golden Gate cloning. (BBa\_K2670000 = pSB1C3\_p35S-46\_LexA, BBa\_K2670001= pSB1C3\_uGFP)

On Tuesday, Ulrich met Iris Richter, an expert in agricultural law and drones law.

Doris started the ethanol receptor system in *P.pastoris* a third time and we got pretty good results.

We also started measuring the toggle switch combined with liposome absorption to test if it can switch ON or OFF.

PCR for Laccase (Anneal Tm = 72°)

PCR for mutated Laccase (Anneal Tm = 72°)

[![BB3 Laccase](https://static.igem.org/mediawiki/2018/a/a4/T--BOKU-Vienna--2018_pic22.jpg)](https://2018.igem.org/Team%3ABOKU-Vienna/pic22.jpg)

1. Ladder
2. Laccase
3. Laccase
4. Laccase
5. Mut. Laccase
6. Mut. Laccase
7. Mut. Laccase
8. Laccase

This figure shows a restriction analysis of BB1\_Laccase & BB1 mut.Laccase. The same parts we also sent to sequencing.


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

# Team:BOKU-Vienna/Experiments

## Experiments

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

# Team:BOKU-Vienna/Results

## Results