iGEM 2017 — Team NAWI_Graz

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# Team:NAWI Graz/Notebook

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# Notebook

The notebook contains a brief summary of everything we did in the course of the competition, starting from the lab safety training at the beginning of the project to finalizing our Wiki at the end of October. In addition to working in the laboratory, we did not only do several team building activities, but also organized many different public events to make genetic engineering more popular and to raise awareness for the iGEM competition.

**Week 1: 3rd of July - 9th of July**

The iGEM team [TUDelft](https://2017.igem.org/Team%3ATUDelft/Collaborate) organized an european iGEM Meet-up - the team of Graz, of course, could not be missing. We sent a [delegation](https://2017.igem.org/Team%3ANAWI_Graz/Collaborations) to join the meet-up. Interesting presentations about synthetic biology and talks with other iGEM teams, were a good start into a summer of work and offered us the opportunity to form collaborations.

![](https://static.igem.org/mediawiki/2017/6/6d/Notebok1.png)

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**Week 2: 10rd of July - 16th of July**

The real journey started in july, when we decided that team building would be a good opportunity to start motivated into a summer of lab-work.
That is why we made a trip to a so-called "Buschenschank", where you can enjoy very good self-made food and drinks.
As the days went on, motivation increased and everyone felt they could meet the challenges ahead.
During the first week, we took part in a safety training about good laboratory practice – correct waste disposal, clean and decent laboratory work. In addition to this lab safety training, we further had a safety seminar where all people who worked at the institute participated. There we got used to all institute related safety questions. Both safety seminars took place before officially starting with the lab work.

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**Week 3: 17rd of July - 23th of July**

Due to our work in the lab, we had one person who was in charge for the safety precaution. Our [“safety authorized person”](https://2017.igem.org/Team%3ANAWI_Graz/Safety)organized a training for all team members including fire drill and evacuation, how to behave in case of an accident and how to render first aid.
Slowly, everyone felt comfortable working in the lab. Since we were working in a large building, during the first weeks, it was hard to find all the different rooms for the various devices.
Besides this, we were also very happy to welcome our new lab buddy “thymio” - an open-source robot from EPFL University.
At this point, we want to thank all our [sponsors](https://2017.igem.org/Team%3ANAWI_Graz/Attributions) , who supported us with lab material and chemical substances.
Last but not least, to celebrate the end of a busy semester, we organized a [“Spritzerstandl”](https://2017.igem.org/Team%3ANAWI_Graz/Engagement) for all friends of iGEM. As a special surprise, we offered 3D-printed bacteriophages and microscopes made by a team colleague.

![](https://static.igem.org/mediawiki/2017/a/a2/Notebook7.jpg)

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**Week 4: 24rd of July - 30th of July**

Finally, our primers and gBlocks from IDT arrived. We diluted a couple of primers - in fact many, many primers. After a while, all primers were ready for PCR---the first step of successful cloning. Also, the biobricks we needed, provided by iGEM, were extracted from the plates and transformed into *E. coli* to duplicate them. Sounds easy, but *E. coli* is not always willing to absorb plasmid DNA. After some tries, we finally convinced *E. coli* to duplicate our gBlocks and biobricks. The next and last step for this week was our Miniprep-session, to get our biobricks and gBlocks in higher concentrations. Lab-work during the weekend can be exhausting, but the results of this hard work were worth it.

![](https://static.igem.org/mediawiki/2017/0/03/Notebook10.jpg)

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**Week 5: 31rd of July - 6th of August**

This week was a very exciting one!
Our robot was put into operation for the first time, a lot of work was done to install the electronics and to test the setup for the robot, the bioreactor and the arena. After finishing this, we tried to get the construction started. Shortly afterwards we achieved a success - a milestone was reached!
In week 4, we also started the interlab study and additionally, we got a new load of laboratory materials from our sponsors for further lab work - thanks again!

![](https://static.igem.org/mediawiki/2017/c/c3/Notebook15.jpg)

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**Week 6: 7rd of August - 13th of August**

This week we performed an endless amount of PCRs - PCR-party :-) - using Q5-polymerase with proof-reading activity from NEB (NewEnglandBiolabs) in order to prepare our DNA-fragments for cloning. We also had an extended 5-hour-meeting to discuss the progressing temperature-sensing-project. The fluorescence-measurement chamber could finally distinguish between active and inactive bacteria and we worked on comparing the data received from the reactor setup to measurements with a spectrophotometer.
An important step for collaborations was the skype conference with the iGEM team of Chennai, India, where we talked about M9 media and its implications for bacteria cultivation when using the alkaline-induced alx-promoter.
Moreover, we decided to establish wednesdays as weekly pizza-days and some of us enjoyed nature while hiking at Rettenbachklamm in Graz.

![](https://static.igem.org/mediawiki/2017/d/d2/Notebook18.jpg)

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**Week 7: 14rd of August - 20th of August**

After many oePCRs to make Gibson-cloning more efficient, we unboxed our HiFi-Assembly Kit sponsored by NEB to perform said Gibson-cloning. Special thanks to NEB for providing great chemically competent *E.coli* DH5α cells. Not having to make competent cells ourselves saved a lot of time. *E.coli* DH5α grew really well on our LB-agar plates. To make sure that only *E.coli* cells with our plasmid can survive on the agar-plates, we added chloramphenicol. And actually we got many clones, which needed to be tested. This very “enjoyable” pipetting work was shifted to the following week.
In the meantime, we tried our first demonstration of the robot in the arena using the temperature-sensitive bacteria we had received.

![](https://static.igem.org/mediawiki/2017/1/1f/Notebook22.jpg)

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**Week 8: 21rd of August - 27th of August**

This week was extremely intriguing, we examined our transformation-plates from Gibson-cloning for positive clones. For this purpose, a colonyPCR was carried out. This sounds like lots of pipetting work and it definitely is. Our main actor in this PCR-reaction is the DreamTaq-polymerase, but also many other ingredients need to be added for a successful PCR, like dNTPs, primers, the templates to test, and, of course, the polymerase-buffer to ensure that the polymerase feels comfortable during amplifying the DNA.
And behold---we got positive clones in the form of bands in the right size on agarose gel! These positive clones were cultivated over night and the plasmid DNA was isolated by Miniprep. Finally, the DNA was ready to be sent for sequencing to “Microsynth”, which was so nice to offer us a lot of free sequencing labels. Slowly the pH-construct got ready for expression!
Meanwhile, the robot in the laboratory was already running fast (although controlled by the bacteria reacting to heat-shock fluorescence proteins that have been provided to us).

![](https://static.igem.org/mediawiki/2017/4/4c/Notebook25.jpg)

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**Week 9: 28rd of September - 9th of September**

Week eight was a rather successful one. The pH-project finally showed first results - we more or less finished cloning our constructs - and we were able to start planning the experiments for the pH-dependent expression of fluorescence protein.
Additionally, we pretty much finished our work on the temperature-inducible expression of GFP with the heat shock promoter ibpA.
On another front, a valuable team member left us for his semester abroad in South Korea. Starting this week, he will be performing mainly assignments that can be done over the internet. We’ll meet again at the Giant Jamboree in Boston.

![](https://static.igem.org/mediawiki/2017/7/75/Notebook29.jpg)

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**Week 10: 4rd of September - 9th of September**

Expression of the pH-construct posed some major challenges. First of all, buffering capacity of the M9 media made it difficult to change the pH-values in the bacterial cultures accordingly. Somehow a solution for our problems with pH-dependent expression must be found. For that reason we had a never ending meeting until late at night,
which was very challenging, but in the end we managed to find a solution.
To make lab-work more fun again, we started drawing with *E. coli* on agar-plates harboring differently colored fluorescent proteins.
Additionally, we filmed a video describing our project to be able to start a crowdfunding campaign.

![](https://static.igem.org/mediawiki/2017/6/6b/Notebook31.jpg)

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**Week 11: 11rd of September - 17th of September**

This week we had a great opportunity to present ourselves and the iGEM project to new students at the Welcome Days organised by the TU Graz. This event is a three day long presentation of all general bachelor study opportunities at the Technical University of Graz together with representatives of all bigger student project groups. As a special surprise we tempted visitors with special petri shell pudding to our information booth. Really delicious! In addition, we send our alx-promotor to our collaboration partners -[SVCE\_Chennai](%E2%80%9Dhttps%3A//2017.igem.org/Team%3ASVCE_CHENNAI%E2%80%9D) in India. The expression with alx & asr made some nice progress too.

![](https://static.igem.org/mediawiki/2017/b/be/Notebook34.jpg)

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**Week 12: 18rd of September - 24th of September**

After spending the whole summer in the lab and after many events and a lot of pizza-days, the daily-university routine was almost back. Regular courses started again and exams had to be written.
Before studying peaks, we organized an education event: We visited an Austrian High School to talk about iGEM in general, our project idea and synthetic biology. To make it even more understandable for the students on how genetic engineering works, we performed some experiments with them like extracting DNA from strawberries.

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**Week 13: 25rd of September - 1th of October**

After some weeks of cloning, the results of the final cloning-work were outstanding - finally: Biobrick-Submission was the goal.
Attaching prefix and suffix to our biobricks and cloning them into our vector pSB1C3 sounds like simple restriction-cloning. In theory it is an easy (especially due to Snapgene, a great, free programming tool, which helped us during the whole iGEM competition) simulation of a whole restriction-cloning process completed within minutes. Unfortunately, the real practical part in the lab looks a little bit different - every step takes hours and many problems and pitfalls emerge. After many working steps, finally a successful transformation turned up, got cloned and after restriction-digest and ligation process, we finally got clones to test, but it was still far from being finished. Sadly, often after colonyPCR no clones with the biobrick in it were found. Instead, only the vector without insert (biobrick) turned up. We were far from giving up and after a few night shifts in the lab, we finally managed to get our biobricks, which were ready for submission.

![](https://static.igem.org/mediawiki/2017/0/0b/Notebook40.jpg)

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**Week 14: 2rd of October - 8th of October**

A lot of thirsty freshmen had to be served at our last official "Spritzerstandl", this time with “Sturm”, shortly fermented grape must - a local speciality in Austria, nice and sweet tasting wine.
These “Spritzerstandls” are the perfect method to introduce the iGEM competition to new students and other interested people. The first steps to form an the new iGEM team NAWI\_Graz 2018 were made this evening.

![](https://static.igem.org/mediawiki/2017/0/0c/Notebook43.jpg)

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**Week 15: 9th of October - 15th of October**

The Nerd Nite took place! An exciting event for students and people interested in science dealing with serious scientific topics in a humorous way, while the audience enjoys a late-bar feeling and drinking. And, best of all, we heard the best bee jokes ever this night!
(Examples:
What is the favorite TV show of bees? .... Game of drones.
Who are bees afraid of? Honeybal Nector.
Why did the bee leave the hive? It was a claustrophobee!)

![](https://static.igem.org/mediawiki/2017/1/1d/Notebook46.jpg)

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**Week 16: 16rd of October - 22th of October**

We organised a real interesting event at our university. Together with experts in the fiel of biochemistry, medicine, biotechnology, philosophy et cetera, and a representative of our team, we held a public discussion about the general awareness of genetic engineering and synthetic biology in public. We had a quite nice talk about several themes like improving the general knowledge, explaining some legal developments in europe and future applications of genetic engineering. The publicity always had the chance to ask in between and got all their questions answered. In the end we served self-baked cakes and some drinks.

![](https://static.igem.org/mediawiki/2017/2/28/Notebook50.jpg)

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**Week 17: 23rd of October - 29th of October**

Finally, our wiki week started! After a crash course in HTML, all team members were sitting together working on the wiki pages all night long. We spent the last week eating pizza and drinking Cola. Just like real coder.

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# Team:NAWI Graz/Experiments

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# Experiments

Describe the research, experiments, and protocols you used in your iGEM project. These should be detailed enough for another team to repeat your experiments.

Please remember to put all characterization and measurement data for your parts on the corresponding Registry part pages.

##### What should this page contain?

* Protocols
* Experiments
* Documentation of the development of your project

##### Inspiration

* [2014 Colombia](https://2014.igem.org/Team%3AColombia/Protocols)
* [2014 Imperial](https://2014.igem.org/Team%3AImperial/Protocols)
* [2014 Caltech](https://2014.igem.org/Team%3ACaltech/Project/Experiments)

[![](https://static.igem.org/mediawiki/2017/a/ad/BiocraftLab.png)](https://www.biocraftlab.com/)
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[![](https://static.igem.org/mediawiki/2017/4/4b/Bisy.png)](http://www.bisy.at/)
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[![](https://static.igem.org/mediawiki/2017/3/34/Oeh.png)](https://oehunigraz.at/)
[![](https://static.igem.org/mediawiki/2017/3/3d/Microsynth.png)](http://www.microsynth.at/)
[![](https://static.igem.org/mediawiki/2017/d/d4/ServoLab.png)](https://www.laborgeraete-laborbedarf.at/)
[![](https://static.igem.org/mediawiki/2017/7/74/VBKaernten.png)](http://www.volksbank-kaernten.at/)
[![](https://static.igem.org/mediawiki/2017/6/64/GRAZ.png)](https://www.graz.at/)
[![](https://static.igem.org/mediawiki/2017/c/c0/SnapGene.png)](http://www.snapgene.com/)
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# Team:NAWI Graz/Results

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# RESULTS

The following results show that both of our constructs work nearly as expected. The alx-mNeonGreen construct shows, that it can be induced by change from pH 7.0 to pH above 8 and fluorescence was measured 20 minutes after this pH shift. The asr-mCardinal construct was inducible by change from pH 7.0 to pH 5.0 and lower but no fluorescence was detected. The promoter itself was working as expected and we were able to detect the 6xHis-tagged mCardinal protein in cultures at pH 5.0 through a Western Blot.

## pH-Sensing

### Alkaline induced expression

To test if our pH sensitive constructs express the fluorescence proteins, we cultivated the bacteria hosting the plasmids in LPM with pH 7 overnight. For expression control of our alx-mNeonGreen construct, we inoculated 20 ml LPM with pH 7.0, 8.0 and 8.5 to an OD600 of 0.2 using the overnight culture. After 20 and 40 minutes 1 ml of each culture was taken and adjusted to the lowest OD600 of the three samples to standardize the OD600. This was necessary as growth at pH 8.0 and 8.5 was significantly slower than at pH 7.0. The diluted samples were used to measure fluorescence (excitation 490 nm, emission 520 nm) and again OD600 with our plate reader. Three aliquots of each sample were measured (n=3), fluorescence was divided by OD600 and averages of the three aliquots were taken to obtain the data shown in Figure 1.

![[Diagramm_alx.xlsx]](https://static.igem.org/mediawiki/2017/d/dd/Asr_res.png)

**Fig 1. Alx controlled mNeonGreen Expression:** Culture medium was buffered to pH 7, 8 and 8.5 and inoculated to an OD600 of 0.2. After 20 and 40 minutes, aliquots were taken and fluorescence (excitation 490 nm, absorbance 520 nm) and OD600 were measured with the plate reader (n=3).

As shown in Fig. 1 fluorescence increases at pH 8.0 and 8.5 at both times compared to a standard culture at pH 7.0. At pH 7.0 cultures still show high fluorescence values with no difference between cultures with our plasmid or cultures without any DNA coding for mNeonGreen (Data not shown). This leads to the conclusion that bacteria on their own emit at ~520 nm when stimulated at 490 nm. Still fluorescence is significantly increased in all cultures at higher pH with a maximum of fluorescence at pH 8.5 after 20 min. This increase in fluorescence allows to distinguish between induced and uninduced. Hence we could show that our [construct](https://2017.igem.org/Team%3ANAWI_Graz/https%3A//2017.igem.org/Team%3ANAWI_Graz/pHPlasmid#alx) works as planned. The next step would be to test how much base (NaOH) would be needed to change the pH of a defined volume of culture with pH 7.0 to pH 8.5. This data would enable us to use the construct to control the robot. Unfortunately the summer was to short to complete this task.

### Acid induced expression

To test acid induced expression of mCardinal controlled by the asr promoter (our asr-mCardinal construct), we tried the same protocol we used for the alx-mNeonGreen but with pH 7.0, 5.0 and 4.5. Unfortunately this did not work out, because, as we discovered later, expression works, but mCardinal, a fluorescence protein with extreme fast maturation time, is designed for expression in mammalian1 cells and does not show any fluorescence when expressed in *E. coli*. Therefore we investigated the expression on the protein level with Western blotting. To do so we cultivated bacteria containing the construct overnight in LPM with pH 7.0 and used this culture to inoculate 100 ml LPM with pH 7.0, 5.0 and 4.5 to an OD600 of 0.1. The cultures were incubated at 37°C. Before inoculating, after 1.5, 2.0 and 2.5 hours 3 OD units were taken of each culture, pelleted and cooled on ice. After all samples were collected they were prepared for SDS gel electrophoresis by sonicating, mixing with loading buffer and heating to 95°C. Samples and pageruler protein standard were  [separated on a SDS gel and blotted](https://2017.igem.org/Team%3ANAWI_Graz/WP) on a nitrocellulose membrane. After washing and incubating with anti-6xHis antibodies tagged with horse radish peroxidase, detection was performed with Thermo ScientificTM SuperSignalTM West Pico Chemiluminescent Substrate.

![[Diagramm_alx.xlsx]](https://static.igem.org/mediawiki/2017/7/70/Westernblot_asr_Graz.jpeg)

**Fig 2. asr controlled expression of mCardinal:** Overlay of the membrane showing the PageRuler prestained protein ladder (S) and a picture showing the signal of the horse radish peroxidase substrate. Lanes from left to right, protein ladder, inoculation culture with pH 7.0, the three samples, pH 7.0, 5.0, 4.5, after 1.5 h (t1), after 2 h (t2) and after 2.5 h (t3).

All detected signals seen in Fig. 2 are the same height as the 30 kDa band of the standard, correlating with the size of mCardinal plus TEV-site, f-degron and the 6xHis-tag, with an approximate weight of 29.9 kDa. There is no signal in any sample with pH 7.0 leading to the conclusion that asr is not active at pH 7.0. At pH 5.0 and 4.5 there are bands of different intensity at all time points, hence asr is active at these pH and mCardinal is expressed. This is proof that our construct does work. Also expression is higher at pH 5.0 than at 4.5 especially after 2h (t2) and 2.5 h (t3). To verify these results and use the construct for our robot, a different fluorescence protein can be inserted in the construct and the new construct could be tested and used like the alx construct above.

1. Chu, J. et al. Non-invasive intravital imaging of cellular differentiation with a bright red-excitable fluorescent protein. Nature methods 11, 572–578 (2014).

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