iGEM 2022 — Team aboa

{% extends "layout.html" %}
{% block title %}Notebook{% endblock %}
{% block lead %}{% endblock %}
{% block title\_class %}notebook-title{% endblock %}
{% block page\_content %}

From the very beginning, we started to document our iGEM journey in detail. We wrote meeting notes about every team and subteam meeting and put all collaboration meetings on our calendar. This was nice also for all team members since now we can look back together and remember all the great memories. Outside the meetings and other events summarized on this page, there are also countless hours of working hard on our project, which can not be documented in any sensible way. On the left side of the timeline, you can see our general activities, whereas on the right side you can take a closer look at our laboratory experiments.

20.12.2021 Team meeting

Present: Malin, Juuli, Nelli, Minttu, Anni, Jesper, Sini, Evelina, Jonna, Iida, Otto, Kristiina, Kevät

The ABOA 2022 kickoff! Malin and Juuli, who continued from the ABOA 2021 team, introduced the competition, tracks, and the most important deliverables and deadlines.

![A picture of the presentation slide.](https://static.igem.wiki/teams/4207/wiki/aboa-notebook-welcome.png)

JAN

08.01.2022 Team meeting

Present: Malin, Juuli, Nelli, Jonna, Anni, Jesper, Otto, Iida, Sini, Kristiina, Evelina, Kevät, Minttu

The first official team meeting of the year! We discussed practicalities, for example schedules, meeting times, different subteams, and grouping activities. In addition, we started to brainstorm our project topic ideas.

![A picture of the Zoom window.](https://static.igem.wiki/teams/4207/wiki/aboa-notebook-meeting1.png)

12.01.2022 Wellbeing day

We had a game night on Zoom to get to know each other better and have fun together.

15.01.2022 Team meeting

Present: Anni, Evelina, Iida, Jesper, Jonna, Juuli, Kevät, Kristiina, Malin, Minttu, Nelli, Otto, Sini

We decided on subteam members and continued brainstorming activities.

![A picture of the Zoom window.](https://static.igem.wiki/teams/4207/wiki/aboa-notebook-meeting2.png)

17.01.2022 Media & Visual Design meeting

Present: Evelina, Iida, Jonna, Juuli, Kevät, Kristiina, Malin, Minttu, Nelli, Otto, Sini

We decided on subteam roles and discussed the upcoming team photo shooting day, team logo, and social media channel usage.

![A picture of the Zoom view.](https://static.igem.wiki/teams/4207/wiki/aboa-notebook-220117media.png)

19.01.2022 Wet Lab meeting + Brainstorming

Present: Iida, Kevät, Kristiina, Malin, Otto, Jonna, Evelina, Anni, Jesper

Subteam roles were decided and team member availabilities were discussed.

![A picture of the Zoom view.](https://static.igem.wiki/teams/4207/wiki/aboa-notebook-220119wetlab.png)

20.01.2022 Fundraising meeting

Present: Sini, Nelli, Kristiina, Malin, Iida, Juuli, Jonna, Jesper

We decided subteam roles, such as the subteam leader and treasurer, as well as discussed the sponsors for our team clothing and office space.

![A picture of the Zoom view.](https://static.igem.wiki/teams/4207/wiki/aboa-notebook-220120fundraising.png)

22.01.2022 Team meeting

Present: Anni, Evelina, Iida, Jesper, Jonna, Juuli, Kevät, Malin, Minttu, Nelli, Otto, Sini

We discussed things decided on previous week’s subteam meetings and our Aboa Turku ry organization. In addition, brainstorming activities were continued.

![A picture of the Zoom window.](https://static.igem.wiki/teams/4207/wiki/aboa-notebook-meeting3.png)

25.01.2022 Human Practices meeting

Present: Anni, Jesper, Jonna, Juuli, Kevät, Kristiina, Malin, Nelli, Otto

We divided different roles and discussed which kinds of things human practices actually include.

![A picture of the Zoom view.](https://static.igem.wiki/teams/4207/wiki/aboa-notebook-220125hp.png)

26.01.2022 Fundraising workshop + Brainstorming

We helped each other out in writing fundraising emails and reaching possible sponsors.

29.01.2022 Team meeting

Present: Anni, Evelina, Jesper, Juuli, Kevät, Kristiina, Malin, Minttu, Nelli, Otto, Sini

Human Practices criteria were introduced and we discussed the possible activities regarding it. We also discussed other subteam updates and planned upcoming photography sessions.

![A picture of the Zoom window.](https://static.igem.wiki/teams/4207/wiki/aboa-notebook-meeting4.png)

31.01.2022 Fundraising meeting

Present: Evelina, Iida, Jonna, Juuli, Kristiina, Malin, Nelli, Sini

We discussed more our partnership packages and what to offer to sponsors.

![A picture of the Zoom view.](https://static.igem.wiki/teams/4207/wiki/aboa-notebook-220131fundraising.png)

FEB

01.02.2022 Human Practices meeting + Brainstorming

Present: Anni, Jesper, Jonna, Juuli, Kevät, Kristiina, Malin, Nelli, Otto

The main theme of this subteam meeting was to discuss values and prepare a presentation of them for our next team meeting.

![A picture of the Zoom view.](https://static.igem.wiki/teams/4207/wiki/aboa-notebook-220201hp.png)

02.02.2022 Media meeting + Meeting with our PI

### Media meeting

Present: Evelina, Jonna, Juuli, Minttu, Nelli, Sini

We planned our social media posts and discussed the situation with our own web page content.

![A picture of the Zoom view.](https://static.igem.wiki/teams/4207/wiki/aboa-notebook-220202media.png)

### Meeting with PI

We presented our current topic ideas to our PI and got valuable feedback from him.

03.02.2022 Visual Design meeting + Brainstorming

Present: Iida, Minttu, Nelli, Otto, Sini

We planned visual aspects of our team photos, such as clothing, location, and placement. The color palette of the team was also designed.

![A picture of the Zoom view.](https://static.igem.wiki/teams/4207/wiki/aboa-notebook-220203vd.png)

05.02.2022 Team meeting

Present: Anni, Evelina, Jonna, Kristiina, Malin, Minttu, Nelli, Otto, Sini

We discussed our fundraising activities and paying the competition fee. The office negotiations are ongoing and brainstorming is still continuing. Team values were also discussed.

![A picture of the Zoom window.](https://static.igem.wiki/teams/4207/wiki/aboa-notebook-meeting5.png)

08.02.2022 Brainstorming

11.02.2022 IT meeting + Brainstorming

Present: Iida, Juuli, Minttu, Sini, Malin

We divided subteam roles and discussed the situation of our own web page.

![A picture of the Zoom view.](https://static.igem.wiki/teams/4207/wiki/aboa-notebook-220211it.png)

12.02.2022 Brainstorming

13.02.2022 Team meeting + Photography session

Present: Anni, Evelina, Iida, Jesper, Jonna, Juuli, Kristiina, Malin, Minttu, Nelli, Otto, Sini

We discussed the situation with brainstorming and voted on new logo colors. We also took team photos and different photos of our team members.

15.02.2022 Presentation at the Plant Biology Seminar

![People standing in the front of a lecture hall giving a presentation.](https://static.igem.wiki/teams/4207/wiki/wiki-communication-plantbiologyseminar.jpg)

19.02.2022 Team meeting

Present: Evelina, Iida, Jesper, Jonna, Malin, Minttu, Nelli, Sini

Subteam activities and progress were discussed.

![A picture of the Zoom window.](https://static.igem.wiki/teams/4207/wiki/aboa-notebook-meeting7.png)

21.02.2022 Fundraising meeting

Present: Evelina, Iida, Jesper, Jonna, Kristiina, Malin, Nelli, Sini

We updated each other on the situation with sponsors, team clothing, and office space, as well as started to plan one grant application.

![A picture of the Zoom view.](https://static.igem.wiki/teams/4207/wiki/aboa-notebook-220221fundraising.png)

22.02.2022 Brainstorming

23.02.2022 Team meeting

Present: Evelina, Iida, Jesper, Jonna, Kevät, Kristiina, Malin, Minttu. Otto, Sini

We continued brainstorming on our project topic and discussed the team clothing.

![A picture of the Zoom window.](https://static.igem.wiki/teams/4207/wiki/aboa-notebook-meeting8.png)

24.02.2022 Sponsor meeting

Present: Malin, Nelli, Sini

We had a meeting with the staff of the University of Turku regarding the financial support for our competition fee.

27.02.2022 Brainstorming

MAR

02.03.2022 Fundraising meeting

Present: Iida, Jesper, Jonna, Malin, Nelli, Sini

We discussed recent progress on different aspects of fundraising, such as sponsors, team clothing, grant applications, and office space.

![A picture of the Zoom view.](https://static.igem.wiki/teams/4207/wiki/aboa-notebook-220302fundraising.png)

03.03.2022 Team meeting

Present: Anni, Jesper, Jonna, Kristiina, Malin, Minttu, Nelli, Otto, Sini

Discussions on study credits had progressed and we got our proposal of getting credits for doing iGEM accepted. Brainstorming activities were also discussed and our final topic idea of plant disease detection came up for the first time. In addition, we went through the iGEM awards and other things that need to be kept in mind.

![A picture of the Zoom window.](https://static.igem.wiki/teams/4207/wiki/aboa-notebook-meeting9.png)

06.03.2022 Brainstorming

09.03.2022 Team meeting

Present: Anni, Evelina, Iida, Jesper, Juuli, Nelli, Otto, Sini

We discussed how to start trying to get more sponsors and closed the deal about our office and meeting spaces. Practicalities, such as meeting times and places, and communication channels were decided.

![A picture of the Zoom window.](https://static.igem.wiki/teams/4207/wiki/aboa-notebook-meeting10.png)

10.03.2022 Brainstorming

14.03.2022 Media meeting

Present: Evelina, Jonna, Juuli, Malin, Minttu, Sini

Once again, we discussed possible social media content, web page content updates, and possible press contacts.

![A picture of the Zoom view.](https://static.igem.wiki/teams/4207/wiki/aboa-notebook-220314media.png)

16.03.2022 Team meeting

Present: Anni, Jesper, Jonna, Juuli, Kevät, Malin, Sini

Updates from different subteams were discussed.

![A picture of the Zoom window.](https://static.igem.wiki/teams/4207/wiki/aboa-notebook-meeting11.png)

18.03.2022 Brainstorming

22.03.2022 Human Practices meeting

Present: Anni, Jesper, Jonna, Kevät, Kristiina, Nelli, Otto

This week’s discussion topics included the organizing of wellbeing day, the sustainability of team clothing, and the ethics related to current project topic ideas.

![A picture of the Zoom view.](https://static.igem.wiki/teams/4207/wiki/aboa-notebook-220322hp.png)

23.03.2022 Team meeting + Fundraising meeting

### Team meeting

Present: Anni, Iida, Jesper, Juuli, Kevät, Malin, Nelli, Otto

Subteam updates, team clothing, and communication channels were discussed further.

![A picture of the Zoom window.](https://static.igem.wiki/teams/4207/wiki/aboa-notebook-meeting12.png)

### Fundraising meeting

Present: Iida, Jesper, Juuli, Nelli

We updated each other on recent fundraising-related things and shared some responsibilities.

![A picture of the Zoom view.](https://static.igem.wiki/teams/4207/wiki/aboa-notebook-220323fundraising.png)

29.03.2022 Sponsor meeting + School visit

A lecture about synthetic biology -related things and iGEM was given for high school students.

30.03.2022 Team meeting + school visit

### Team meeting

Present: Anni, Evelina, Jonna, Juuli, Kevät, Kristiina, Malin, Minttu, Nelli, Otto, Sini

We pitched two current project topic ideas, the detection of potato/plant viruses and the detection of borreliosis, to each other. We voted and decided to go further with the topic of potato/plant viruses. Possible future collaborations and sustainable team clothing options were also covered.

![A picture of the Zoom window.](https://static.igem.wiki/teams/4207/wiki/aboa-notebook-meeting13.png)

### School visit

Another lecture about similar topics was given, but for different target groups in the same high school.

APR

04.04.2022 Wellbeing day

We had a relaxed evening and played board games together.

![People playing board games and card games.](https://static.igem.wiki/teams/4207/wiki/aboa-notebook-boardgames.jpg)

05.04.2022 Fundraising meeting + Wet Lab meeting

### Fundraising meeting

Present: Nelli, Malin, Jesper, Kristiina, Jonna, Juuli, Sini, Iida

The main theme of this subteam meeting was to discuss recent progress on getting sponsors, and how we could improve and boost it.

![A picture of the Zoom view.](https://static.igem.wiki/teams/4207/wiki/aboa-notebook-220405fundraising.png)

### Wet Lab meeting

Present: Iida, Kristiina, Malin, Otto, Jonna, Anni, Jesper

We discussed project design, including a possible dry lab module, our research plan, lab protocols, and reagent orders.

![A picture of the Zoom view.](https://static.igem.wiki/teams/4207/wiki/aboa-notebook-220405wetlab.png)

06.04.2022 Team meeting

Present: Iida, Malin, Minttu, Nelli

This team meeting consisted of brief updates on recent things.

08.04.2022 Human Practices meeting

Present: Jesper, Jonna, Juuli, Kevät, Otto

We continued discussions about the sustainability of team clothing options as well as updated our team values based on the feedback we got.

![A picture of the Zoom view.](https://static.igem.wiki/teams/4207/wiki/aboa-notebook-220408hp.png)

12.04.2022 Meeting with Aachen

13.04.2022 Team meeting

Present: Malin, Nelli

We discussed recent subteam activities.

14.04.2022 Wet Lab meeting

Present: Kevät, Kristiina, Malin, Jonna, Evelina, Anni, Jesper

The main discussion topics in this meeting were crude extract preparation, expression construct generation, and cell-free reaction mixture preparation.

![A picture of the Zoom view.](https://static.igem.wiki/teams/4207/wiki/aboa-notebook-220414wetlab.png)

20.04.2022 Wet Lab meeting + Human Practices meeting

### Wet Lab meeting

Present: Kevät, Kristiina, Malin, Otto, Jesper

The project design and project novelty options were discussed and we decided to contact people who have done previous work in the same field.

![A picture of the Zoom view.](https://static.igem.wiki/teams/4207/wiki/aboa-notebook-220420wetlab.png)

### Human Practices meeting

Present: Jesper, Jonna, Juuli, Kevät, Kristiina, Malin, Nelli, Otto

We discussed some collaboration opportunities and how to proceed with them.

![A picture of the Zoom view.](https://static.igem.wiki/teams/4207/wiki/aboa-notebook-220420hp.png)

22.04.2022 Visual Design meeting + Fundraising meeting

### Visual Design meeting

Present: Juuli, Minttu, Nelli, Otto

We started the planning of the promotion video and the next team photo.

![A picture of the Zoom view.](https://static.igem.wiki/teams/4207/wiki/aboa-notebook-220422vd.png)

### Fundraising meeting

Present: Iida, Jesper, Juuli, Malin, Nelli

Once again, we discussed possible ways to get more sponsors and money.

![A picture of the Zoom view.](https://static.igem.wiki/teams/4207/wiki/aboa-notebook-220422fundraising.png)

27.04.2022 Team meeting

Present: Iida, Jesper, Juuli, Malin, Nelli, Otto

Possible collaborations with other iGEM teams and other recent updates were discussed.

![A picture of the Zoom window.](https://static.igem.wiki/teams/4207/wiki/aboa-notebook-meeting16.png)

28.04.2022 Wet Lab meeting

Present: Iida, Kevät, Kristiina, Malin, Otto, Jonna, Jesper

We discussed some recent issues with our project, such as difficulties in finding sequences.

![A picture of the Zoom view.](https://static.igem.wiki/teams/4207/wiki/aboa-notebook-220428wetlab.png)

MAY

02.05.2022 Meeting with Aachen + Expert meeting

03.05.2022 Fundraising workshop

We wrote grant applications and emails to possible sponsors together.

04.05.2022 Team meeting + Wet Lab meeting

### Team meeting

Present: Iida, Jesper, Juuli, Kevät, Kristiina, Minttu, Nelli

Collaborations and other updates were discussed. The special prizes were also introduced and we started to consider achieving them.

![A picture of the Zoom window.](https://static.igem.wiki/teams/4207/wiki/aboa-notebook-meeting17.png)

### Wet Lab meeting

Present: Iida, Kevät, Kristiina, Jonna, Jesper

We updated each other on recent discussions with other experts and how we could modify our project with the help of the information that we got.

![A picture of the Zoom view.](https://static.igem.wiki/teams/4207/wiki/aboa-notebook-220504wetlab.png)

09.05.2022 Visual Design meeting

Present: Iida, Juuli, Minttu, Nelli

We continued the planning of our promotion video and wrote a preliminary storyline for it.

11.05.2022 Team meeting + Fundraising workshop

Present: Jesper, Juuli, Kristiina, Malin, Minttu, Nelli, Otto

In this week’s team meeting, we discussed problems in the wet lab and possible activities regarding our integrated human practices work. In addition, we went through this year’s medal criteria and decided on which gold medal criteria we are trying to fulfill.

18.05.2022 Team meeting + Wellbeing day

Present: Anni, Iida, Jesper, Jonna, Juuli, Kevät, Kristiina, Malin, Minttu, Nelli

Discussions about different collaboration and partnership opportunities were done. Other than that, we had no major things to discuss. After the meeting, we went to the sauna together.

![A picture of the Zoom window.](https://static.igem.wiki/teams/4207/wiki/aboa-notebook-meeting19.png)

20.05.2022 Human Practices meeting

Present: Jesper, Kevät, Kristiina, Malin, Otto ( + Yasoo Morimoto)

The main discussion topics included integrated human practices activities such as interviewing farmers and other stakeholders.

24.05.2022 Wet Lab meeting

Present: Kevät, Kristiina, Malin, Jonna, Evelina, Jesper (+ Michael Burgis)

We got a visitor to this meeting, iGEM alumni Michael Burgis, who provided us with tips on finding novelty factors and on overall wet lab design. In addition, the meeting included a lot of troubleshooting.

![A picture of the Zoom view.](https://static.igem.wiki/teams/4207/wiki/aboa-notebook-220524wetlab.png)

25.05.2022 Team meeting

Present: Anni, Iida, Jesper, Kevät, Kristiina, Malin

We updated each other about things going on in different subteams and also discussed more the gold medal criteria we aim to achieve.

![A picture of the Zoom window.](https://static.igem.wiki/teams/4207/wiki/aboa-notebook-meeting20.png)

JUN

01.06.2022 Team meeting + Media meeting

### Team meeting

Present: Anni, Jonna, Juuli, Minttu, Nelli

We discussed wiki writing and decided to start writing now. In addition, collaborations with different iGEM teams were covered.

![A picture of the Zoom window.](https://static.igem.wiki/teams/4207/wiki/aboa-notebook-meeting21.png)

### Media meeting

Present: Anni, Jonna, Juuli, Minttu, Nelli

We discussed our social media content related to the Education & Communication gold medal criterion and sponsor agreements.

![A picture of the Zoom view.](https://static.igem.wiki/teams/4207/wiki/aboa-notebook-220601media.png)

02.06.2022 Meeting with Toronto

![A picture of the Zoom view.](https://static.igem.wiki/teams/4207/wiki/aboa-notebook-toronto.png)

06.06.2022 Meeting with Tirupati

08.06.2022 Team meeting + Meeting with Aalto-Helsinki

Present: Anni, Jesper, Jonna, Juuli, Kevät, Malin, Nelli

This time, we discussed a lot about our current and possible future collaborations with other iGEM teams. In addition, the wet lab subteam introduced their current situation and progress.

![A picture of the Zoom window.](https://static.igem.wiki/teams/4207/wiki/aboa-notebook-meeting22.png)

09.06.2022 Media meeting + Wet Lab meeting

### Media meeting

Present: Jonna, Juuli, Minttu, Nelli

We planned the topics of our educational posts for different social media channels.

### Wet Lab meeting

Present: Kevät, Malin, Jonna, Anni, Jesper

We updated the current situation with the whole project as well as discussed how to modify our project based on the information we got from recent expert interviews.

![A picture of the Zoom view.](https://static.igem.wiki/teams/4207/wiki/aboa-notebook-220609wetlab.png)

10.06.2022 Wellbeing day

13.06.2022 Media meeting + Meeting with TrigGate

### Media meeting

Present: Anni, Evelina, Juuli, Minttu

We planned how to reveal our project topic on different social media channels.

### Meeting with TrigGate

![A picture of the Zoom view.](https://static.igem.wiki/teams/4207/wiki/aboa-notebook-220613triggate.png)

14.06.2022 Meeting with Uppsala

15.06.2022 Team meeting

Present: Anni, Jesper, Jonna, Juuli, Kevät, Malin, Minttu, Nelli, Sofia

Our team meeting started with introducing our new team member, Sofia. She will attend the wet lab subteam. Other than that, every subteam presented their work and progress to other members.

![A picture of the Zoom window.](https://static.igem.wiki/teams/4207/wiki/aboa-notebook-meeting23.png)

16.06.2022 Expert meeting

17.06.2022 LBA plates, cultures and restriction analysis for ZikaV sensor and trigger

Present: Kevät, Jesper

**Growth plates**
(LBA, 20 ml)

2x LBA+Amp 100 µg/ml

1x LBA+Kan 500 µg/ml

Streaked ZikV-trigger (Amp) and ZikV-sensor (Amp) to LBA-plates, started incubation at 37℃ at 3 PM. The plates were moved to the fridge at 8:01 AM

Single colonies were inoculated from both plates to culture tubes containing 2xYT-medium with the aforementioned antibiotics. Incubation started at 16.30 and moved to the fridge at 8.47.

875 µl from each of the cultures were used to make a 10 % glyserol preps which are stored at -80℃. The plasmids were purified from the rest of the cultures (3225 µl) with Monarch Plasmid Miniprep Kit. The purified plasmids are stored at -20℃. The concentration of the purified plasmids were measured with biodrop.

The plasmids were confirmed to contain the inserts by restriction analysis. Zikv-Trigger was cut with AvrII and Zikv-Sensor with EcoRI in a 25 µl reaction. They were incubated at 37℃ for 4 hours, followed by 80℃ inactivation for 20 min and then kept at 4℃.

19.06.2022 Media meeting

Present: Anni, Jesper, Jonna, Juuli, Minttu, Nelli

We finished some educational posts together and finalized the plan about how to reveal our topic.

20.06.2022 LBA plates, preparing liquid media YT and YT+P, tetracycline stock, cultures for ZikaV sensor and trigger

Present: Kristiina, Kevät, Jesper

**Cell cultures**

Three bottom up tubes were taken from the laboratory. 4 ml of 2 x YT was added into each of the tubes in a laminar flow cabinet. 4 µl of ampicillin (stock c = 100 mg/ml) was added into two of the tubes, 4 µl of kanamycin (stock c = 50 mg/ml) was added into one of the tubes. Streaks were taken separately from the following plates: 1) AMP100 pAD-lyseR 2) AMP100 ZikaV-Trigger 3) KAN50 Zika-V-sensor. Now we have three different cultures: one with a streak from AMP100 pAD-lyseR and ampicillin, AMP100 ZikaV-Trigger and ampicillin and Kan50 ZikaV-Sensor and kanamycin. Cell cultures were grown overnight at 200 rpm at 37 ℃.

**Tetracycline stock + LBA plate**

Tetracycline stock was created by weighing 13.3 mg of tetracycline to 1.33 ml of UP water. LBA plate was created by adding 25 µl of tetracycline stock to 25 ml of melted LBA, plating 20 ml of it.

LBA + T plate was streaked with bacteria from Addgene stab.

**YT + YTP**

2 x 1 L bottles of 2xYT were created by weighing 16 g tryptone, 10 g yeast extract and 5 g NaCl to a bottle and filling to 1 L.

4 x 1L bottles of 2xYTP were created as above, with the addition of 3.99 g of potassium phosphate monobasic and 13.93 g of potassium phosphate dibasic.

**4 x LBA + AMP100 µg/m**

About 90 ml of LBA and 90 µl of ampicillin (stock c = 100 mg/ml) was added into a glass bottle. Shaked delicately. Poured onto four plates.

**4 x LBA + KAN100 µg/ml**

About 90 ml of LBA and 180 µl of kanamycin (stock c = 50 mg/ml) was added into a glass bottle. Shaked delicately. Poured onto four plates.

**1 x LBA + TET10 µg/ml**

About 20 ml of LBA and 20 µl of tetracycline (stock c = 10 mg/ml) was added into a glass bottle. Shaked delicately. Poured onto a plate.

21.06.2022 Plasmid extraction and digestion for ZikaV sensor and trigger, more LBA plates

Present: Evelina, Kristiina, Jesper

***E. coli* ZikaV + *E. coli* ZikaS Plasmid extraction**

Plasmids were extracted with the Monarch Plasmid Mini Prep Kit. The pellet was centrifuged down with 2800 x g, 5 min, +4°C. Rest of the steps were centrifuged as described in the protocol (approx. 16 000 x g).

The DNA concentration of the plasmid extract was determined using BioDrop. The results were as follows: ZikaV-Sensor 279.2 ng/µl; ZikaV-Trigger 136.0 ng/µl.

**ZikaV-S and ZikaV-T plasmid digestion**

Plasmid digestion was made from the extracted ZikaV-S and ZikaV-T plasmids for gel electrophoresis to determine that the extraction was successful. The following reactions were prepared:

|  | ZikaV-S | ZikaV-T |
| --- | --- | --- |
| DNA, 1 µg | 3,58 µl | 7,35 µl |
| rCutSmart Buffer | 5 µl | 5 µl |
| H20 (MQ) | 40,42 µl | 36,65 µl |
| Restriction enzyme | 1 µl (EcoRI-HF) | 1 µl (AvrII) |
|  | 50 µl | 50 µl |

Glyserol preps were made of the ZikaV-Sensor and ZikaV-Trigger cell cultures for the cell/plasmid library. The preps were stored in -80℃ in 2B/Patrikainen no.:s 146 and 147.

**Cell cultures**

Three bottom up tubes were taken from the laboratory. 4 ml of 2 x YT was added into each of the tubes in a laminar flow cabinet. 4 µl of ampicillin (stock c = 100 mg/ml) was added into two of the tubes, 4 µl of kanamycin (stock c = 50 mg/ml) was added into one of the tubes. Streaks were taken separately from the following plates: 1) AMP100 pAD-lyseR 2) AMP100 ZikaV-Trigger 3) KAN50 Zika-V-sensor. Now we have three different cultures: one with a streak from AMP100 pAD-lyseR and ampicillin, AMP100 ZikaV-Trigger and ampicillin and Kan50 ZikaV-Sensor and kanamycin. Cell cultures were grown overnight at 180 rpm at 37°C.

**1 x LBA**

Approximately 20 ml of LBA was poured onto a plate.

22.06.2022 Gel electrophoresis for digested ZikaV sensor and trigger plasmids, 2XYTPG preparation

Present: Kristiina, Kevät, Jesper

**Gel Electrophoresis of ZikaV-S and ZikaV-T plasmid digestions**

The gel was made following the Gel Electrophoresis Protocol in our Drive: 0.6 g Agarose, 60 ml 1XTAE, and 6 µl SYBR Safe. The digestion samples were split in two so in total four samples were made: 2X ZikaV-S digested by EcoRI-HF and 2X ZikaV-T digested by AvrII. The samples were prepared as follows: 25 µl DNA from the digestion and 5 µl Loading Dye Purple 6X (NEB). The ladder used was Purple 1 kb Plus DNA Ladder (NEB).

The gel was run for 37 min on 100 V.

![A picture of  agarose gel electrophoresis for digested plasmids. One band can be seen in all four sample wells.](https://static.igem.wiki/teams/4207/wiki/220622-gel.png)

**Figure 1.**
Agarose gel electrophoresis for digested plasmids. Gel diagram: 1: Ladder 2: ZikaV-S-1 3: ZikaV-S-2 4: ZikaV-T-1 5: ZikaV-T-2.

The samples seem to be inverted, as the ZikaV-S plasmid is supposed to be 6455 bp and ZikaV-T plasmid 5675 bp but on the gel, ZikaV-T seems to be bigger than ZikaV-S. New digestion & gel will be made tomorrow to determine that the fault is due to misnaming the samples while digestion and that the extracted plasmids are named correctly and good to use.

**2XYTPG preparation**

400 ml of 2xYTPG was prepared by dissolving 7.2 g of glucose to 100 ml of 2xYTP and filter sterilizing it. Then, 300 ml of sterile 2xYTP was added.

22.06.2022 Team meeting

Present: Anni, Iida, Jesper, Juuli, Kevät, Kristiina, Malin, Nelli, Otto

Besides other updates, we discussed getting more iGEM teams from Finland. That ideation process had already been started by the previous ABOA team, and we definitely want to continue from it! In addition, we voted for the project name from various different options and ended up with CropFold!

![People on a picnic.](https://static.igem.wiki/teams/4207/wiki/aboa-notebook-picnic.jpg)

23.06.2022 ZikaV sensor and trigger plasmid extraction, digestion and electrophoresis, starter culture for BL21-Gold-dLac

Present: Evelina, Kristiina, Kevät, Jesper

**ZikaV S and ZikaV T Plasmid digestion**

Following reactions were prepared:

| ZikaV Sensor | ZikaV Trigger | Negative control (H2O) |
| --- | --- | --- |
| DNA 3.6 µl | DNA 7.4 µl\* | - |
| rCutSmart 5 µl | rCutSmart 5 µl | rCutSmart 5 µl |
| EcoRI-HF 1.0 µl | AvrII 1.0 µl | EcoRI-HF 10 µl |
| H2O 40.4 µl | H2O 36.5 µl | H2O 44 µl |

\* Accidentally might have added 7.14 µl instead of 7.4 µl.

Reactions were incubated at 37°C for 4 hours, then 20 minutes at 80°C and kept at 4°C for ∞.

**Gel Electrophoresis of ZikaV-S and ZikaV-T plasmid digestions**

The gel was made following the Gel Electrophoresis Protocol in our Drive: 0.6 g Agarose, 60 ml 1XTAE, and 6 µl SYBR Safe. The digestion samples were split in two so in total four samples were made: 2X ZikaV-S digested by EcoRI-HF and 2X ZikaV-T digested by AvrII. The samples were prepared as follows: 25 µl DNA from the digestion and 5 µl Loading Dye Purple 6X (NEB). The ladder used was Purple 1 kb Plus DNA Ladder (NEB).

The gel was run for 40 min on 100 V.

![A picture of  agarose gel electrophoresis for digested plasmids. One band can be seen in all four sample wells.](https://static.igem.wiki/teams/4207/wiki/220623-gel.png)

**Figure 2.**
Agarose gel electrophoresis for digested plasmids. Gel diagram: 1: Ladder 2: ZikaV-S-1 3: ZikaV-S-2 4: ZikaV-T-1 5: ZikaV-T-2 6: Negative control H2O.

Now the bands are about the right size (both about 6 kb and the ZikaV-S is bigger than ZikaV-T) and also neg control doesn't show any band --> everything should be alright.

**Plasmid extraction**

Extracted plasmids from yesterday's cultures with Monarch Plasmid miniprep (NEB). This time the last resuspension was made with 50 µl of the elution buffer instead of 30 µl.

Determined the DNA concentration with BioDrop. ZikaV-T: 87.24 ng/µl; ZikaV-S: 85.28 ng/µl.

**Starter culture**

4 ml of 2xYT was supplemented with 4 µl of tetracycline and a single BL21-Gold-dLac colony from plate was inoculated o/n.

24.06.2022 Cell lysis sonication, production culture for autolysis, Bradford assay

Present: Evelina, Kristiina, Jesper

**Cell lysis by sonication**

Sonicated the samples from yesterday. 4 cycles of 30 s sonication and 40 s rest. Peak value 12 and samples were on ice for the whole time. Centrifuged at 20 000 g, 4°C for 60 minutes to pellet the cell debris.

**Determined the amount of protein with Bradford assay.**

Made the standard curve with a stock solution of 1 mg/ml of BSA. Made the following dilutions: 100 µg/ml, 10-1 µg/ml. Only used 2, 4, 6, 8, 10 µg/ml dilutions for the standard curve. Measured the absorption at 595 nm.

**Autolysis production culture**

Starter culture was inoculated into 400 ml of 2xYTPG with 100 µg/ml concentration of Ampicillin (Amp). Incubation at 37℃, 220 rpm was started at 8.40

**BL21-Gold-dLac**

1L flask containing 400 ml of 2xYTPG was supplemented with 100 µg/ml Amp and 4 ml of starter culture. Incubation was started at 8.40.

OD600 was measured at 1 hr intervals, using 2xYTPG as a blank. OD-value of 6.08 was reached at 14.40. Culture was supplemented with 2.2 ml of 0.2 M IPTG and incubated at RT until 17.41.

The culture was transferred to 10 50 ml falcons, each having 40 ml of culture. They were centrifuged at 1800 g, RT for 15 min. Supernatant was discarded and each pellet was resuspended in 4.5 ml of cold S30A buffer. Then they were combined into one falcon tube and centrifuged as above. Then, supernatant was discarded and pellet was weighed. Pellet mass: 1.66 g. Pellet was resuspended in 3320 µl of cold S30A and was divided into 3 eppendorf tubes before freezing at -80℃.

27.06.2022 Autolysis concentration measurement, preparation of S30A

Present: Kristiina, Kevät, Otto, Jesper

Suspension was thawed at RT water bath. Then it was vortexed, transferred to a culture tube, vortexed for 3 min and incubated at 37℃, 300 rpm, starting at 9.45. At 10.34 the suspension was vortexed for 3 min, before starting incubation again at 10.39. At 11.27 the tubes were put on ice, transferred to eppendorfs and centrifuged at 20 000 g, 4℃ for 90 min.

During centrifuging, 0-10 µg/ml BSA standard was prepared and stardard curve was created. The concentration of the lysate was determined from 1/10000 dilution, it was 13.42 mg/ml. Concentration was < 27 mg/ml, so Amicon Ultra-4 10K concentrator was used to concentrate the solution. New concentration was determined from 1/10000 and 1/20000 dilutions and it was 83.2 mg/ml. The solution was diluted to 30.7 mg/ml by diluting 182 µl of the solution with 311.8 µl of water. Then, solution was aliquoted into 30 µl aliquots and stored at -80℃.

**S30A**

Prepared 1 L of S30A, pH 7.69 to be autoclaved tomorrow.

27.06.2022 Human Practices meeting

Present: Jesper, Juuli, Kevät, Kristiina, Nelli

We familiarized ourselves more with the gold medal criterion Education & Communication and planned how to achieve it. We also updated our team values and decided to collaborate with Aalto-Helsinki by giving workshops for children in Heureka, the Finnish science center.

![A picture of the Zoom view.](https://static.igem.wiki/teams/4207/wiki/aboa-notebook-220627hp.png)

28.06.2022 Starter culture for BL21 Gold deLac, preparation of S30B, 2xYTPG, 1M DTT

Present: Kristiina, Kevät, Otto, Jesper

**S30B pH 8.20**

Prepared 2 L of S30B, pH 8.20.

**2 X YTPG**

Prepared 660 ml of 2xYTPG with 11.88g of glucose and filtered it.

**1 M DTT**

Prepared 6 ml of 1M DTT and filtered it, but due to filtering there're only 5 mls of it anymore....

**BL21 Gold deLac starter cultures**

Made 2x 4.5ml BL21-Gold-dLac starter cultures with 4.5 µl 10 mg/ml tetracycline. 37°C in a shaker from 16:05 onwards.

29.06.2022 Energy mix components preparation, production culture for sonication, preparation of Tris

Present: Kevät, Otto, Jesper

**Energy mix - components**

HEPES 2M

4.766 g of HEPES was diluted in approximately 7 ml of MQ-water. After that solution was titrated to reach pH 8.02 with KOH and filled until volume V= 10 ml. Stored at -20°C.

GTP 75mM

ATP 75mM

tRNA 50 mg/ml

5 mg of tRNA was weighed to an Eppendorf tube. 100 µl of MQ-water was added. Mixed gently with a pipette. Stored at -80°C.

NAD 175mM

0.0117 g NAD was weighted to a PCR tube and diluted in 91 µl of MQ-water. After dilution 9 µl of 2M Tris was added to adjust pH around 8 (from Arce et al.). Mixed gently and stored at -20°C.

CoA 65mM

0.0049 g CoA was weighted to a PCR tube and diluted in 100 µl of MQ-water. MIxed gently and stored at -20°C.

cAMP 650mM

0.0118 g cAMP was weighted to a PCR tube and diluted in 31.7 µl of MQ-water. After dilution 18.3 μl of 2M Tris was added to adjust pH around 7.5-8 (from Arce et al.). Mixed gently and stored at -20°C.

Folinic acid 33.9mM

Spermidine 1M

Maltodextrin 650 mg/ml

2.6 g of maltodextrin was diluted in 2.5 ml of MQ-water in a 15 ml Falcon tube. After dilution 0.65 g of maltodextrin was added and filled until V = 5 ml. Stored at 4°C.

HMP 0.03 g/ml

0.12 g of HMP was diluted in 2 ml of MQ-water in a 15 ml Falcon tube and filled until V = 4 ml. Incubated at +95°C for 5 minutes. Stored at -80°C.

**Production culture**

2 L flasks with 330 ml 2xYTPG each were inoculated with 3.3 ml of the overnight culture and 330 µl of Tet was added to each flask. Incubation at 37°C, 220 rpm was started at 9.00. Cultures reached 0.606 and 0.634 OD600 at 12.20. 1.65 ml 0.2 M IPTG was added to each flask. Incubation was continued at RT, 220 rpm until 15.25.

After the IPTG incubation, culture was divided equally to 2 500 ml centrifuge bottles and centrifuged at 5000 g, 4℃ for 12 min. Supernatant was discarded and pellet was suspended in 100 ml of S30A and centrifuged as above. This step was repeated. Pellets were resuspended with 20 ml of S30A per bottle and then transferred to a falcon tube, which was centrifuged at 2000 g, 4℃ for 8 min. Supernatant was discarded and wash repeated with 40 ml of S30A. Then the pellet was centrifuged for 2 min and resuspended in 4 ml of S30A. The suspension was stored at -20℃.

**Tris**

Approximately 36.342 g of Tris base was diluted in 150 ml of MQ-water. The concentration of the solution is 2 M. The solution was filtrated with a syringe and a filter.

**Glycerol prep of *E. coli* lyseR**

0.875 µl of *E. coli* lyseR was added into a microcentrifuge tube and 0.125 µl of 80 % glycerol was added. Mixed with a pipette. Stored at -80°C.

29.06.2022 Team meeting + Wet Lab meeting

### Team meeting

Present: Anni, Iida, Jesper, Jonna, Juuli, Kevät, Minttu, Nelli

We discussed the work that different subteams have done during the past week.

![A picture of the Zoom window.](https://static.igem.wiki/teams/4207/wiki/aboa-notebook-meeting25.png)

### Wet Lab meeting

Present: Iida, Kevät, Jonna, Evelina, Anni, Jesper, Juuli, Nelli

The work done during the past weeks was discussed and problems with sonication and cell cultures were troubleshot. The writing of the laboratory notebook was also recapped so that everyone is aware of the requirements related to it.

![A picture of the Zoom view.](https://static.igem.wiki/teams/4207/wiki/aboa-notebook-220629wetlab.png)

30.06.2022 Culture for BL21 pET-15 lacZ, transformation for pEAS002 (mScarlet-I), Primer dilutions for mScarlet and lacZ

Present: Kevät, Otto, Jesper

**5 X LBA+KAN50 Plates**

Approximately 120 ml of melted LBA was into a flask. 120 µl of kanamycin (stock concentration 50 mg/ml) was added. Mixed. Poured onto six plates. One plate failed.

**LacZ culture**

BL21 (DE3) strain containing the pET-15b-LacZ-plasmid was streaked to an LBA + Amp 100 plate. Incubation at 37℃ started at 10.03. Single colonies from the plate were inoculated into a 5 ml 2xYT + Amp 100 culture tube and incubation was started at 20.20.

**2 x Liquid cultures of *E. coli* BL21 pET-15 lacZ**

5 ml of 2 x YT was transferred into a tube in a laminar flow cabinet. 5 µl of ampicillin (stock concentration = 100 mg/ml) was added. Mixed. A streak of a plate containing *E. coli* BL21 pET-15 lacZ was added. Two liquid cultures were prepared. Grown overnight at 37°C at 250 rpm.

**mScarlet transformation**

2 preps of competent DH5a cells were thawed on ice for 15 min. 1 µl of Purified pEAS001 (mScarlet) and 1 µl of pEAS002 (mScarlet-I) were added to the tubes. They were incubated on ice for 30 min. Cells were heat-shocked on 42℃ water bath for 45 s and kept on ice for 5 min. Then, 500 µl of LB was added to each of the tubes and they were placed on 37℃, 200 rpm for 1 hr. From each of the tubes, 100 µl was plated on a LBA + Kan 50 plate and the remaining 400 µl on a second plate. They were incubated at 37℃, starting at 16.05.

**Primer dilutions for mScarlet and lacZ**

**100 µM dilutions:**

mScarlet F

n = 30.8 nmol

V = n/c => 30.8 ⋅ 10
-9
/ 100 ⋅ 10
-6
M = 0.000 308 l = 308 µl

308 µl of MQ-water was added into the tube containing 31.6 nmol mScarlet F. Mixed. Stored at -20°C.

mScarlet R

n = 31.6 nmol

V = 31.6 ⋅ 10
-9
/ 100 ⋅ 10
-6
M = 0.000 316 l = 316 µl

Prepared the same way as mScarlet F except for the volume of MQ-water.

lacZ F

n = 25.2 nmol

V = 25.2 ⋅ 10
-9
mol/ 100 ⋅ 10
-6
M = 0.000 252 l = 252 µl

Prepared the same way as mScarlet F except for the volume of MQ-water.

lacZ R

n = 35.2 nmol

V = 35.2 ⋅ 10
-9
mol/ 100 ⋅ 10
-6
M = 0.000 352 l = 352 µl

Prepared the same way as mScarlet F except for the volume of MQ-water.

**10 µM dilutions:**

180 µl of MQ-water was added into four microcentrifuge tubes. 20 µl of each primer (c = 100 µM) was added into separate tubes. Stored at -20°C. Total amount: four different tubes for mScarlet F and R, lacZ F and R

30.06.2022 Meeting with Tirupati

JUL

01.07.2022 PCR for LacZ, sonication and Bradford assay for sonicated cells

Present: Evelina, Kevät, Otto, Jesper

**Sonication**

Pellet suspension was thawed on ice and then sonicated for 4x30 s. Suspension was centrifuged at 48 000 g, 4℃ for 30 min. Supernatant was transferred to 2 15 ml falcons and incubated at 37℃, 220 rpm for 60 min.

Solution was transferred to eppendorfs and centrifuged at 12 000 g, 4℃ for 10 min. 10 µl of the supernatant was saved for Bradford assay. The rest was loaded into a 10 ml 10 MWCO dialysis cassette. Dialysis was completed in a 1 L beaker at 4℃ for 3 hours.

**Bradford assay**

BSA standards were created at 0, 2, 4, 6, 8 and 10 µg/ml concentrations, 800 µl of each were created and 200 µl of Coomassie solution was added to each. Their absorbance at 595 nm was measured and a standard plot was created. Sample was diluted to 1/10000 and 1/20000 for the assay. The concentration of the sample undiluted sample was 41,01 mg/ml.

**LacZ PCR**

lacZ PCR amplification from plasmid

Following reaction was prepared:

| Volume (Vtot = 25 µl) | Reagent | Concentration |
| --- | --- | --- |
| 1.25 µl | lacZ F primer | 0.5 µM |
| 1.25 µl | lacZ R primer | 0.5 µM |
| 1 µl | pET-15 lacZ Plasmid DNA | 30.77 ng |
| 12.5 µl | Q5 High-Fidelity 2 X MasterMix | 1 X |
| 12.5 µl | Q5 High-Fidelity 2 X MasterMix | 1 X |
| 9 µl | MQ-Water | - |

Following PCR cycle was run:

|  |  |  |
| --- | --- | --- |
| **Initial Denaturation** | 98°C | 30 s |
| **1) Denaturation** | 98°C | 10 s |
| **2) Annealing** | 59.13°C | 30 s |
| **3) Extension** | 72°C | 30 s |
| **Final Extension** | 72°C | 30 s |
| **Hold** | 4°C | ∞ |

Steps 1)-3) repeated 40 X. Stored at -20°C.

04.07.2022 Gel electrophoresis and extraction LacZ PCR products, preparation of competent DH5a cells and transformation for LacZ plasmid, minipreps for mScarlets

Present: Anni, Kevät, Jesper

**One-day competent DH5a cells**

Competent cells were made using the modified protocol for competent cells (fast) from Hari's (SBC group) protocol. DH5α cells from LBA plate inoculated into 2 x 8 ml SOC in 25 ml Erlenmeyers. Incubation at 37 ℃ and 220 rpm until cells reached OD600 value of 0.4-0.5. The cells were then centrifuged at 10000 x g, 4 ℃, 2min.

**lacZ cleanup and concentration**

lacZ was cleaned up with Monarch PCR & DNA Cleanup Kit (5 µg) Protocol Card according to the DNA Cleanup and Concentration (because lacZ gene size is 3075 bp > 50 bp). Sample type of lacZ is dsDNA < 2 kb (some amplicons, fragments) so 5:1 was chosen as the ratio of binding buffer:sample (125 µl:25 µl). The concentration of the purified lacZ sample was 62.97 ng/µl.

**Transformation to DH5α cells with lacZ-plasmid**

Transformation was made using the heat-shock transformation protocol for E. coli cells. 10 µl DNA (lacZ plasmids) on the cells. Cells were incubated on ice little over than half an hour (maybe 35 min) → heat shock 45 s 42 ℃ → kept 6 min on ice. 1 hour 10 min incubation at 37 ℃. We used kanamycin as antibiotics. We plated 100µl on LBA plate and the remaining cell culture on another LBA plate.

**Agarose gel electrophoresis for lacZ**

60 ml of 1 x TAE buffer was heated with 0,60 g agarose. 6 µl of SYBR safe color was added and mixed. Left to cool. Poured onto gel tray. Approximately 3.75 µl of Gel Loading Dye 6X was added to the sample (sample volume 18 µl). 10 µl of 1 kb Quick-Load DNA Plus Ladder Purple was loaded onto the gel. Approximately 22 µl (all of the sample) was loaded onto the gel. Gel was run at 100 V for 35 mins.

![A picture of gel electrophoresis. Sample well shows three faint bands, and a lot of background.](https://static.igem.wiki/teams/4207/wiki/220704-gel.png)

**Figure 3.**
Gel electrophoresis for LacZ.

**Gel extraction lacZ**

m(lacZ gel slice) = 0.09 g = 90 mg (~ 90 µl)

lacZ gel slice was extracted with Monarch DNA Gel Extraction Kit. The concentration of the purified lacZ was 8.039 ng/µl (quite low!!). Stored at -20°C.

**Minipreps for mScarlets**

Minipreps for mScarlets were made using single colonies of DH5α pEAS001 and DH5α pEAS002 plates. Colonies were inoculated in 5 ml 2xYT + 50 µg/ml Kan.

04.07.2022 Meeting with Patras

05.07.2022 Plasmid extraction for pEA5001 and pEA5002 (mScarlet and mScarlet-I), two PCRs for mScarlet and mScarlet-I and gel electrophoresis, energy mix preparation, RNA synthesis and purification for ZikaV trigger.

Present: Evelina, Jonna, Anni, Kristiina, Kevät, Jesper

**Energy mix**

The reagents were added in order to microcentrifuge tube and Vortex after each addition. The tube was kept on ice. The mix was then dived in tubes, 4 x 150 µl and 1 x 100 µl and put to -80 ℃.

Reagents for energy mix:

| **Reagent** | c | V (µl) |
| --- | --- | --- |
| HEPES | 2 M | 177 |
| ATP+GTP | 75 nm / 56 nm | 141 |
| GTP | 100 nm | 26 |
| CTP | 100 nm | 63 |
| UTP | 100 nm | 63 |
| tRNA | 50 mg/ml | 28 |
| CoA | 65 mM | 28 |
| NAD | 175 mM | 8.08 |
| folinic acid | 33.9 mM | 14 |
| spermidine | 1 mM | 7 |
| maltodextrin | 650 mg/ml | 131 |
|  |  | total 700 µl |

**RNA synthesis of Zika V trigger**

To the reaction tube, the reagents were added in the following order: 1.5 µl 10 X RNAP reaction buffer; 1.5 µl each of the NTPs: ATP (100 mM), GTP (100 mM), CTP (100 mM), UTP (100 mM); 1 µg, 11 µl, Zika V trigger DNA (87.24 ng/µl), 1.5 µl T7 RNAP. 20 µl reaction. The reaction was incubated at 37℃ for 3 hours. Note: After 3 hours of incubation, it was heated to 42℃ for 1 min and incubated at 37℃ for 1 hour.

**RNA pufication**

RNA was purified with Monarch total RNA miniprep kit. 2 volumes (20 µl) of RNA lysis buffer was added to the 20 µl reaction and mixed with pipette. 60 µl of 95 % ethanol was added and mixed with pipette. The mixture was transferred to an RNA purification column and spinned for 30 seconds, the flow-through was discarded. 500 µl of RNA Priming Buffer was added and spinned for 30 seconds, the flow-trough was discarded. 500 µl of RNA Wash Buffer was added and spinned for 30 seconds, the flow-through was discarded. Another 500 µl of RNA was added and spinned for 2 minutes. The column was transferred to a new microcentrifuge tube. The RNA was eluted with 50 µl of water and spinned for 30 seconds. The tube was put to -80℃.

After purification, the RNA concentration was 148.5 ng/µl or about 5.25 mM (calculated without terminator).

**Minipreps for mScarlets and lacZ**

Minipreps for mScarlets were made using single colonies of DH5α pEAS001 and DH5α pEAS002 plates. Colonies were inoculated in 5 ml 2xYT + 50 µg/ml Kan. These mScarlet minipreps are for preparing glycerol preps. Miniprep for lacZ was made using a single colony of DH5α pET-15b lacZ plate. Colony was inoculated in 5 ml 2 x YT + 100 µg/ml Amp. Grown overnight at 220 rpm at 37 ℃.

**Purify mScarlet plasmids**

mScarlet and mScarlet-I plasmids, pEA5001 and pEA5002 respectively, were purified using the NEB plasmid miniprep kit. The first centrifuge step was done as follows: 5 mins, 3000 rcf. This worked well. The plasmids were eluted to 50 µl of the elution buffer.

The concentrations of the DNA in the samples were determined by using BioDrop. pEA5001 61,28 ng/µl; pEA5002 74,68 ng/µl. 2 µl of both samples were used for this, and the rest (48 µl) are in the freezer.

**PCR for both mScarlets**

First PCR for mScarlet and mScarlet-I.

**Second PCR (different temperature) for both mScarlets**

Same amounts as previous one, different temperatures in PCR.

--> failed, PCR machine error (W27), we will try again tomorrow.

**Agarose gel**

Agarose gel was made for mScarlets PCR products. 60 ml of 1 x TAE buffer was heated with 0.60 g agarose. 6 µl of SYBR safe color was added and mixed. Left to cool. Later stored in plastic wrap in the fridge.

05.07.2022 Media meeting

Present: Anni, Juuli, Minttu

We divided responsibilities regarding educational social media content and planned our special challenge for the other iGEM teams in honor of World Emoji Day.

![A picture of the Zoom view.](https://static.igem.wiki/teams/4207/wiki/aboa-notebook-220705media.png)

06.07.2022 PCR, gel electrophoresis and extraction for mScarlet and mScarlet-I, pOdd1 transformation (acceptor plasmid backbone).

Present: Evelina, Anni, Kristiina, Kevät, Otto, Jesper

**Third PCR (different temperature) for mScarlets**

PCR for pEA5001 and pEA5002. Same amounts as yesterday, same temperature (annealing at 72℃) as yesterday's second PCR. The PCR cycle was adjusted!! 2 x 72℃ 30 s was changed to 1 x 72℃ 1 min. The PCR worked just fine but we need to monitor the PCR in the future if there are some problems again.

**Electrophoresis for the mScarlets PCR products**

Agarose gel from yesterday was taken out of the fridge and used in gel electrophoresis.

Samples: 25 µl of each PCR product was mixed with 5 µl of Purple Dye and injected to the wells. Also 7 µl of ladder was injected to two wells. 100 V was used to run the gel for 42 minutes first and then 15 minutes to be sure.

Below is the picture of the gel electrophoresis after 42 min of running.

![A picture of agarose gel electrophoresis.. Wells 2 and 3 both show one bright band. Water control well is empty. Sample wells 7 and 8 both show one bright band.](https://static.igem.wiki/teams/4207/wiki/220706-gel.png)

**Figure 4.**
Agarose gel electrophoresis for mScarlet and mScarlet-I products. 1: Ladder 2: pEA5001, annealing 62 ℃ 3: pEA5002, annealing 62 ℃ 4: H2O, annealing 62 ℃ 5: empty 6: ladder 7: pEA5001, annealing 72 ℃ 8: pEA5002, annealing 72 ℃ 9: H2O, annealing 72 ℃

Gel analysis did show that there is no visible difference between the PCR products (annealing temperature on the left 62℃ and on the right 72℃) so all of the samples were taken to the next step.

**Gel extraction**

Gel extraction was made using Monarch® DNA Gel Extraction Kit protocol from NEB. Wanted DNA fragments were excised out from the gel with sterile knife (remember to use flame and paper to clean the knife) and transferred to 1.5 ml microfuge tubes and weighted (empty microfuge tube to tare):

pEA5001 62°C --> 200 mg --> 800 µl gel dissolving buffer

pEA5002 62°C --> 160 mg --> 640 µl gel dissolving buffer

pEA5001 72°C --> 150 mg --> 600 µl gel dissolving buffer

pEA5002 72°C --> 170 mg --> 680 µl gel dissolving buffer

concentrations of the samples, biodrop ng/µl:

pEA5001 62°C : 58.54 ng/µl

pEA5002 62°C : 35.75 ng/µl

pEA5001 72°C : 56.16 ng/µl

pEA5002 72°C : 44.77 ng/µl

--> put in the freezer.

**Glycerolpreps**

Three glycerolpreps were made: HD5α pET 15b-lacZ, HD5α pEA 5001 and HD5α pEA 5002

--> 875 µl of each sample was mixed with 125 µl glycerol and stored in the freezer -80°C.

**pJUMP sfGFP transformation (acceptor plasmid backbone)**

Transformation was made using the heat-shock transformation protocol for *E. coli* cells.

5 µl DNA (pJUMP sfGFP plasmids) on the cells. Cells were incubated on ice for half an hour, then heat shock 45 s 42℃ and kept 4 min on ice. 500 µl of LB was added and incubated for 1 hour at 37℃. With 200 rpm shaking. We used kanamycin as antibiotics. We plated 100 µl on LBA plate and 400 µl cell culture on another LBA plate.

06.07.2022 Team meeting

Present: Anni, Jesper, Jonna, Juuli, Kevät, Kristiina, Malin, Minttu, Nelli, Otto

In this week’s team meeting, we discussed, among other things, organizing a crowdfunding campaign and Aalto-Helsinki’s upcoming visit to Turku. We also wrapped up our current collaborations with different teams and pondered their effects on our project.

![A picture of the Zoom window.](https://static.igem.wiki/teams/4207/wiki/aboa-notebook-meeting26.png)

07.07.2022 Cell-free reaction with ZikaV, plasmid extractions, PCR, gel electrophoresis and extraction for LacZ, mScarlet and mScarlet-I, gel electrophoresis for ZikaV trigger RNA, LBA plates.

Present: Evelina, Anni, Kristiina, Kevät, Jesper

**Agarose gel and electrophoresis for RNA**

Gel electrophoresis for ZikaV Trigger RNA pure. Before gel electrophoresis the RNA concentration was measured with biodrop (148.5 ng/µl).

Agarose gel was made for the purified Zika V trigger RNA. 60 ml of 1 x TAE buffer was heated with 0.72 g agarose. 6 µl of SYBR safe color was added and mixed. Left to cool.

Sample: 18 µl formamide, 5 µl loading dye, 7 µl Zika V trigger RNA sample = 30 µl. Also 7 µl of ladder was loaded.

100 V was used to run the gel for 10 minutes. Then the voltage was raised by ten after every ten minutes. 110 V after 10 min, 120 V after 20 min, 130 V after 30 min, 140 V after 40 min, 150 V after 50 min. The gel was run for 70 minutes.

![A picture of agarose gel electrophoresis.  Sample wells 2 and 3 show two bands, sample well 3 only one band. Well 5, water control shows some faint bands. Sample wells 7-9 show a clear band, as well as sample wells 11-13.](https://static.igem.wiki/teams/4207/wiki/220707-gel.png)

**Figure 5.**
Agarose gel electrophoresis for ZikaV trigger RNA. Wells: 1. Ladder 2. RNA sample

--> The gel run failed, next time incubation after formamide needs to be performed before loading the sample to the well.

**13 x LBA+KAN50 plates**

Approximately 250 ml of melted LBA was into a flask. 250 µl of kanamycin (stock concentration 50 mg/ml) was added. Mixed. Poured onto 13 plates. LBA might have been too hot when the antibiotic was applied.

**Plasmid extraction for the remaining cultures of mScarlet, mScarlet-I and LacZ.**

Plasmid extraction was done following the instructions of the kit (NEB Monarch Plasmid miniprep). In the first step the culture tubes were centrifuged in 3000 x g for 5 min. In the last step plasmids were eluted to 50 µl of the elution buffer.

The concentrations of the plasmids were: pET-15b 209.8 ng/µl; pEA5001 81.47 ng/µl; pEA5002 80.62 ng/µl.

**PCR reactions for LacZ, mScarlet and mScarlet-I**

3 reactions were made of each sequence (3x LacZ, 3x mScarlet, 3x mScarlet-I); in total 9 reactions + 2 controls with MQ H2O. One of the H2O controls was made using lacZ primers and the other one with mScarlet primers. Assembled as follows:

|  |  |  |
| --- | --- | --- |
| 1.25 µl | forward primer | LacZ f or mScarlet f (same for both mScarlets) |
| 1.25 µl | reverse primer | LacZ r or mScarlet r (same for both mScarlets) |
| 1.25 µl | DNA | pET-15b-lacZ for lacZ, pEA5001 for mScarlet, pEA5002 for mScarlet-I, MQ H2O for controls |
| 12.5 µl | Q5 HighFidelity 2x MasterMix |  |
| 9.0 µl | MQ H2O |  |

The amount of DNA was 25 µl more than the initial amount, because of a mistake in pipetting. This shouldn’t affect the reaction too much, it might slightly increase the amount of the product.

The PCR was run with the following protocol:

|  |  |  |
| --- | --- | --- |
| 98°C | 30 s |  |
| 98°C | 10 s | This step was cycled 40x |
| 72°C | 1 min | This step was cycled 40x |
| 72°C | 2 min |  |
| 4°C | ∞ |  |

**Gel electrophoresis for lacZ, mScarlet and mScarlet-i PCR products**

25 µl of each PCR product was mixed with 5 µl of 6 X Purple Dye and injected to the wells. Notion: the volume of PCR products seemed to differ from each other, which might affect the results. 7 µl of both ladders were injected into two separate wells. The gel was run at 100 V for 35 min.

![A picture of agarose gel electrophoresis. No bands can be seen in any of the wells.](https://static.igem.wiki/teams/4207/wiki/220707-gel-2.png)

**Figure 6.**
Agarose gel electrophoresis for LacZ, mScarlet and mScarlet-I PCR products. Wells: Quick-Load Purple 1 kb DNA Ladder, lacZ 1, lacZ 2, lacZ 3, H2O with lacZ primers, 100 bp Ladder, 001 1 (mScarlet 1), 001 2 (mScarlet 2), 001 3 (mScarlet 3), H2O with mScarlet primers, 002 1 (mScarlet-I 1), 002 2 (mScarlet-I 2), 002 3 (mScarlet-I 3)

**Gel extraction for lacZ, mScarlet and mScarlet-I**

Despite the apparent problems with the PCR, the products were extracted with NEB Monarch DNA Gel extraction kit. The products from the three parallel PCR reactions of each sequence were combined to reduce the amount of the reagents used. LacZ tube 1 and lacZ tube 2 were combined into one lacZ tube (the third was ignored because it didn't produce the correct product); mScarlet 1, 2 and 3 were combined to one mScarlet tube and mScarlet-I 1, 2 and 3 were combined to one mScarlet-I tube.

The weights of the gel pieces in the final tubes: lacZ 80 mg → 320 µl buffer; mScarlet 200 mg → 800 µl buffer; mScarlet-I 220 mg → 880 µl buffer. The samples were eluted to 20 µl in the final step.

The concentrations were:

lacZ 20.60 ng/µl

mScarlet 104.0 ng/µl

mScarlet-I 83.51 ng/µl

**Cell-free reaction 1 for ZikaV**

A Mastermix was prepared using the excel sheet to calculate the amounts to pipette for 90 µl mastermix. Sonication 2/41 was used as the extract. The pipetted amounts are here:

|  |  |
| --- | --- |
| Water | 11.36 µl |
| Amino Acid mix | 18 µl |
| HMP | 1.8 µl |
| K-G | 1.44 µl |
| Mg-G | 1.33 µl |
| ONPG | 7.5 µl |
| PEG | 4.5 µl |
| Cell extract | 32.9 µl |

Mastermix was created in tube 1. From the mastermix, 19.5 µl was transferred to tube 3 (water control) and 0.5 µl of water was added to complete the sample.

0.33 µl of Zika-S plasmid was added to tube 1. 34.1 µl of this mixture was transferred to tube 2 (negative sample). The sample was completed with 0.57 µl of water.

0,57 µl of Zika-T plasmid was added to tube 1 (positive sample)

From tube 3, one 5 µl and one 10 µl sample was loaded to a clear V-bottom 96 well-plate. From tubes 1 and 2, two 20 µl and two 10 µl samples were loaded into the plate as follows:

|  |  |  |  |  |  |  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 | 11 | 12 |
|  |  |  |  |  |  |  |  |  |  |  |  |
|  | 3 | 2 | 2 | 1 | 1 |  |  |  |  |  |  |
|  | 3 | 2 | 2 | 1 | 1 |  |  |  |  |  |  |
|  |  |  |  |  |  |  |  |  |  |  |  |
|  |  |  |  |  |  |  |  |  |  |  |  |
|  |  |  |  |  |  |  |  |  |  |  |  |
|  |  |  |  |  |  |  |  |  |  |  |  |
|  |  |  |  |  |  |  |  |  |  |  |  |

The reaction was incubated at 29℃ for 2 hrs. And additional 1 hour at RT in the beginning, we did not program the reader correctly. Absorbance at 420 nm was measured at 1 min intervals.

![A graph of absorbance measured in cell-free reaction as a function of time with different samples.](https://static.igem.wiki/teams/4207/wiki/220707-cell-free.png)

**Figure 7.**
β-galactosidase activity in cell-free reaction.

07.07.2022 Meeting with Uppsala

08.07.2022 Level one acceptor plasmid pOdd-1 transformation, gel electrophoresis for ZikaV trigger RNA.

Present: Evelina, Anni, Kristiina, Jesper

**Agarose gel and electrophoresis**

Second gel electrophoresis for ZikaV Trigger RNA pure.

Agarose gel was made for the purified Zika V trigger RNA. 60 ml of 1 x TAE buffer was heated with 0.72 g agarose. 6 µl of SYBR safe color was added and mixed. Left to cool.

Sample 1: 18 µl formamide, 5 µl loading dye with SDS, 7 µl Zika V trigger RNA

Sample 2: 18 µl formamide, 5 µl loading dye without SDS, 7 µl Zika V trigger RNA

sample = 30 µl. Also 7 µl of ladder was loaded.

Before loading the samples to the wells samples were incubated for 5 min at 65°C and left on ice for 5 min.

100 V was used to run the gel for 10 minutes. Then the voltage was raised by ten after every ten minutes. 110 V after 10 min, 120 V after 20 min, 130 V after 30 min, 140 V after 40 min, 150 V after 50 min and 160 V after 60 min. The gel was run for 70 min.

![A picture of agarose gel electrophoresis. Very faint bands can be seen on the top of the gel on both of the sample wells.](https://static.igem.wiki/teams/4207/wiki/220708-gel.png)

**Figure 8.**
Agarose gel electrophoresis for ZikaV trigger RNA. Wells: 2. ladder 3. sample 1 (with SDS) 4. sample 2 (without SDS)

--> Gel run still did not work.

**Level one acceptor plasmid pOdd-1 transformation**

Transformation was made using the heat-shock transformation protocol for *E. coli* cells.

5 µl DNA (pOdd-1 plasmids) on the cells. Cells were incubated on ice for almost 50 min, then heat shock 45 s 42℃ and kept 3 min on ice. 500 µl of LB was added and incubated for 1 hour at 37℃ with 200 rpm shaking. We plated 100 µl on LBA plate and 500 µl cell culture on another LBA plate. We used kanamycin as antibiotics.

09.07.2022 10X PBS buffer preparation

Present: Kristiina, Jonna

**10X PBS**

Tried to prepare 10X PBS, but it didn't work.

The pOdd-1 plates were collected from the incubator at 12:30. They didn't seem too bad even though they were incubated too long and were likely overgrown.

11.07.2022 Cell-free reaction with ZikaV, competent DH5α cells preparation, 10X PBS buffer

Present: Evelina, Kristiina, Kevät

**10X PBS**

Recipe: 100 ml of 10X PBS, 8 g NaCl, 0.2 g KCl, 1.44 g Na
2
HPO
4
, 0.24 g KH
2
PO
4
-> to 100 ml MQ H
2
O

The salts were added to 80 ml of MQ water one at a time while stirring with a magnetic stirrer. Then the pH was adjusted to 7.2 with NaOH and the rest of the water was added using a volumetric flask. Then the buffer was filtered it with .2 µm filter to two 50 ml falcons and stored in +4℃.

Prepared 100 µl of 1X PBS by diluting the 10X buffer and then used the 1X PBS for the ONPG solution.

**Competent DH5α cells**

Cold DH5α cells were inoculated in 5 ml of LB medium in two separate tubes. Grown overnight at 220 rpm at 37℃.

**Cell free reaction for ZikaV with sonication cell extract**

A mastermix was prepared in tube 1. Everything was pipetted according to the table below in that order. Sonication 2/41 was used as the extract.

Reagents for mastermix:

|  |  |
| --- | --- |
| Water | 11.36 µl |
| Amino acid mix | 18 µl |
| HMP | 1.8 µl |
| K-G | 1.44 µl |
| Mg-G | 1.33 µl |
| ONPG | 7.5 µl |
| Cell extract | 32.9 µl |
| PEG | 4.5 µl |

Mix was created in tube 1. From the mix, 19.5 µl was transferred to tube 3 (water control) and 0.5 µl of water was added to complete the sample.

0.33 µl of Zika-Sensor plasmid was added to tube 1. 34.1 µl of this mixture was transferred to tube 2 (negative sample). The sample was completed with 0.57 µl of water.

0.57 µl of Zika-Trigger RNA was added to tube 1 (positive sample).

From tube 3, one 5 µl and one 10 µl sample was loaded to a clear V-bottom 96 well-plate. From tubes 1 and 2, two 5 µl and two 10 µl samples were loaded into the plate as follows:

|  |  |  |  |  |  |  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 | 11 | 12 |
|  |  |  |  |  |  |  |  |  |  |  |  |
|  |  |  |  |  |  | 3 5 µl | 2 5 µl | 2 5 µl | 1 5 µl | 1 5 µl |  |
|  |  |  |  |  |  | 3 10 µl | 2 10 µl | 2 10 µl | 1 10 µl | 1 10 µl |  |
|  |  |  |  |  |  |  |  |  |  |  |  |
|  |  |  |  |  |  |  |  |  |  |  |  |
|  |  |  |  |  |  |  |  |  |  |  |  |
|  |  |  |  |  |  |  |  |  |  |  |  |
|  |  |  |  |  |  |  |  |  |  |  |  |

The reaction was incubated at 29℃ for 3 hrs. Absorbance at 420 nm was measured at 1 min intervals.

![A graph of absorbance measured in cell-free reaction as a function of time with different samples.](https://static.igem.wiki/teams/4207/wiki/220711-cell-free.png)

**Figure 9.**
β-galactosidase activity in cell-free reaction.

11.07.2022 IT meeting + Visual Design meeting

### IT meeting

Present: Iida, Juuli, Minttu

We planned our wiki layout and design.

![A picture of the Zoom view.](https://static.igem.wiki/teams/4207/wiki/aboa-notebook-220711it.png)

### Visual Design meeting

Present: Iida, Juuli, Minttu, Nelli, Otto

The promotion video planning was continued and some responsibilities regarding it were shared. We also designed a few options for the CropFold project logo.

![A picture of the Zoom view.](https://static.igem.wiki/teams/4207/wiki/aboa-notebook-220711vd.png)

12.07.2022 Cell-free reaction with pEAS002, competent DH5α cells preparation, PCR for LacZ

Present: Evelina, Jonna, Kristiina, Kevät, Jesper

Some of our growing cells were transferred to new plates.

![A picture of bacteria in plates. All of the bacterial strains have grown on the plates.](https://static.igem.wiki/teams/4207/wiki/220712-plates.png)

**Figure 10.**
A. pET15b-LacZ in *E.coli* DH5 α. B. pET15b-LacZ in *E.coli* DH5 α, pAD-lyseR, ZikaV trigger and BL21 pET15b-LacZ. C. pEAs002 in *E.coli* DH5 α, pEAs001 in *E.coli* DH5 α and ZikaV sensor. D. pOdd1 in *E.coli* DH5 α., pEAs002 in *E.coli* DH5 α and pEAs001 in *E.coli* DH5 α

**Competent DH5α cells**

About 800 µl from the O/N culture was inoculated into 160 ml of LB. Unfortunately, the competent cells failed because we contaminated the new culture accidentally twice when measuring the OD. Two new cultures were made of the O/N culture by inoculating 20 µl of the old O/N culture into 5 ml of LB. Grown overnight.

Our old DH5a plate had gone bad, so we moved the transformation to tomorrow. We should do a new one tomorrow (12.7.) and always keep two plates of them in the fridge so that if something happens to one of them, we'd have a backup.

**LacZ PCR**

lacZ PCR amplification from plasmid

Following 4 X MasterMix was prepared:

| Volume (Vtot = 100 µl) | Reagent | Concentration |
| --- | --- | --- |
| 5 µl | lacZ F primer | 0,5 µM |
| 5 µl | lacZ R primer | 0,5 µM |
| 4 µl | pET-15 lacZ Plasmid DNA | 8,392 ng |
| 50 µl | Q5 High-Fidelity 2 X MasterMix | 1 X |
| 36 µl | MQ-Water | - |

Following PCR cycle was run 40 cycles:

| Initial Denaturation | 98 ℃ | 30 s |
| --- | --- | --- |
| 1) Denaturation | 98 ℃ | 10 s |
| 2) Annealing | 68 ℃ | 30 s |
| 3) Extension | 72 ℃ | 30 s |
| Final Extension | 72 ℃ | 30 s |
| Hold | 4 ℃ | ∞ |

Steps 1)-3) repeated 40 X. Stored at -20 ℃.

OBS! There were problems again with the PCR programme stopping. This time it said something about the lids being wrong.

**Cell-free reaction with pEAS002 to see if we produce any protein in our reactions**

The needed amounts of reagents were calculated in our sheet, using 50 µl as the final volume. Sonication 2/41 and autolysis 1 batches were used as the extract in the samples.  To tubes numbered 1 (samples), we pipetted the following amounts:

| Reagent | Sonication 2/41 (µl) | Autolysis 1 (µl) |
| --- | --- | --- |
| Water | 10.18 | 3.8 |
| Amino acid mix | 10 | 10 |
| HMP | 1 | 1 |
| K-G | 0.8 | 0.8 |
| Mg-G | 0.74 | 0.74 |
| Energy mix | 5 | 5 |
| PEG | 2.5 | 2.5 |
| IPTG | 0.25 | 0.25 |
| Extract | 18.3 | 24.7 |

After pipetting, 24.4 µl of this mastermix was transferred to tubes labeled 2 (water control), followed by 0.6 µl of water. 0.6 µl of pEAS002 (78.4 ng/µl) was added to the samples.

After that, two 10 µl samples from each tube were pipetted to a flat-bottom. black 96-well plate as follows:

| 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 | 11 | 12 |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
|  |  |  |  |  |  |  |  |  |  |  |  |
|  | Sonic. water | Sonic. water | Sonic. sample | Sonic. sample |  |  |  |  |  |  |  |
|  | Autol. water | Autol. water | Autol. sample | Autol. sample |  |  |  |  |  |  |  |
|  |  |  |  |  |  |  |  |  |  |  |  |
|  |  |  |  |  |  |  |  |  |  |  |  |
|  |  |  |  |  |  |  |  |  |  |  |  |
|  |  |  |  |  |  |  |  |  |  |  |  |
|  |  |  |  |  |  |  |  |  |  |  |  |

The reaction was incubated for 3 hrs in 29℃ and the fluorescence (569/593 nm) was measured at 1 min intervals.

![A graph of fluorescent intensity measured from different samples presented as a function of time.](https://static.igem.wiki/teams/4207/wiki/220712-cell-free.png)

**Figure 11.**
mScarlet-I produced in cell-free reaction.

13.07.2022 Team meeting

Present: Anni, Iida, Jesper, Juuli, Kristiina, Malin, Minttu, Nelli, Otto, Sofia

We decided our competition track and discussed Grand Jamboree fees and how we are going to be able to pay them. Other than that, we had long discussions on our collaborations with other teams as well as each person’s responsibilities regarding medal criteria and wiki texts. In addition, we voted for our project logo.

13.07.2022 Cell-free reaction testing different potassium glutamate concentrations, plasmid extraction for pOdd-1, competent DH5α cells preparation

Present: Evelina, Anni, Kristiina, Kevät, Jesper, Sofia

**Cell-free reaction testing different Potassium Glutamate concentrations**

Using the mastermix calculator, the following mastermix was created into tube labeled 56, using sonication 2/41 batch as the extract. Pipetted as follows:

| Water | 38.7 µl |
| --- | --- |
| AA | 38 µl |
| HMP | 3.8 µl |
| Mg-G | 2.8 µl |
| Energy mix | 19 µl |
| PEG | 9.5 µl |
| IPTG | 0.95 µl |
| Extract | 69.5 µl |

11.5 µl of the mastermix was transferred to tube labelled 0 (water control). 0.5 µl of water was added to complete the control.

4.4 µl of pEAS002 plasmid was added to tube 56.

The mastermix was aliquotted by 21.89 µl to tubes 6, 8, 10, 15, 20, 30, 56.

The following amounts of K-G and water were added to each tube:

| Tube | K-G | Water |
| --- | --- | --- |
| 6 | 0.35 M 0.386 µl | 0.22 µl |
| 8 | 0.35 M 0.514 µl | 0.1 µl |
| 10 | 0.35 M 0.643 µl | 0 µl |
| 15 | 1 M 0.338 µl | 0.268 µl |
| 20 | 1 M 0.5 µl | 0.106 µl |
| 30 | 1.75 M 0.386 µl | 0.22 µl |
| 56 | 3.5 M 0.246 µl | 0.246 µl |

10 µl of water sample and 2 x 10 µl of the samples were loaded onto a black flat-bottom 96 well-plate. The reaction was run at 29 ℃ for 3 hrs. The fluorescence at 569/593 nm was measured at 1 min intervals.

No signal was detected from the reaction, likely due to not vortexing the samples.

![A graph of fluorescent intensity measured from different samples presented as a function of time.](https://static.igem.wiki/teams/4207/wiki/220713-cell-free.png)

**Figure 12.**
mScarlet-I produced in cell-free reaction.

**Glycerol prep of *E. coli* pOdd-1**

0.875 µl of *E. coli* pOdd-1 was added into a microcentrifuge tube and 0.125 µl of 80 % glycerol was added. Mixed with a pipette. Stored at -80℃.

**Plasmid pOdd-1 extraction**

Plasmids were extracted from two cultures, the one was pink as it should but the other one wasn't. Plasmid extraction was done using NEB Monarch Plasmid miniprep following the manufacturer’s protocol. In the first step the culture tubes were centrifuged in 3000 x g for 5 min. In the last step plasmids were eluted to 50 µl of the elution buffer.

The DNA concentrations were measured with BioDrop. The pink culture concentration was 73.92 ng/µl and the other one was 64.88 ng/µl.

**Competent DH5α cells**

From the O/N culture, 0.1 - 0.5% was inoculated into 160ml of LB and grew the cells to OD600= ~0.4-0.5. During the procedure the cells were kept on ice as much as possible. Incubated the culture at 4℃ for 30 minutes. Centrifuged at 5000rpm – 5 mins at 4℃ in 4 x 50 ml Falcon tubes. The centrifuge was maintained at 4℃. The supernatant was discarded. The cells were resuspended in each tube in 20 ml of ice-cold sterile 0.1 M MgCl2 and incubated in ice for 15 mins. The cells were centrifuged at 5000 rpm 4℃ for 5 mins. The supernatant was discarded and resuspended the cells in 25 ml of ice-cold sterile 0.1 M CaCl2 and incubated in ice for 15 mins. The cells were centrifuged (5000 rpm - 5mins - 4℃) and the supernatant was discarded. The cells were resuspended in each tube in 1ml of ice-cold sterile 0.1 M CaCl2 supplemented with 15-20% glycerol and aliquoted 100 µl into each 1.5 ml tube. Stored immediately at -80℃.

**Zika-T RNA production**

To a 200 µl PCR tube, the following reagents were added in the following order. 1.5 µl 10 X RNAP reaction buffer; 1.5 µl each of the NTPs: ATP (100 mM), GTP (100 mM), CTP (100 mM), UTP (100 mM); 1 µg, 11 µl, Zika V trigger DNA (87.24 ng/µl), 1,5 µl T7 RNAP. The final volume was 20 µl. Reaction was incubated in a 37℃ water bath for 5 hrs and the reaction tube was stored at +4℃.

14.07.2022 Wet Lab meeting + Meeting with Patras and TecCEM

### Wet Lab meeting

Present: Kevät, Kristiina, Malin, Jonna, Anni, Jesper, Sofia

The main theme of this meeting was troubleshooting, but we also discussed the progress done in different sectors of the wet lab as well as planned our troubleshooting collaboration with Aalto-Helsinki.

### Meeting with Patras and TecCEM

![A picture of the Zoom view.](https://static.igem.wiki/teams/4207/wiki/aboa-notebook-220714patras.png)

14.07.2022 Testing the cell-free system with 2/41 sonication batch and variable potassium glutamate concentrations, gel electrophoresis for LacZ, 12 transformations, ZikaV trigger RNA synthesis and purification

Present: Evelina, Jonna, Anni, Kristiina, Kevät, Jesper, Sofia

**Testing the cell-free system with 2/41 sonication batch and variable Potassium Glutamate concentrations**

Using the master mix calculator (excluding the water from the calculator), the following master mix was created taking care to vortex each stock and the master mix after each addition. The extract was not vortexed, and neither was the master mix after adding extract. Sonication 2/41 batch was used as the extract. Pipetted as follows:

| AA | 38 µl |
| --- | --- |
| HMP | 3.8 µl |
| Mg-G | 2.8 µl |
| Energy mix | 19 µl |
| PEG | 9.5 µl |
| IPTG | 0.95 µl |
| Extract | 69.5 µl |

10.68 μl of the mastermix was transferred to tube labelled 0 (water control). 1.32 μl of water was added.

17.36 μl if the mastermix was aliquotted to tubes 4, 6, 8 10, 15, 20, 30 and 56. Then they were filled to 22.25 μl with the following amounts of water and Potassium Glutamate:

| Tube | K-G | Water (μl) |
| --- | --- | --- |
| 4 | 0.175 M 0.514 μl | 3.45 |
| 6 | 0.175 M 0.771 μl | 3.2 |
| 8 | 0.175 M 1.03 μl | 2.94 |
| 10 | 0.175 M 1.286 μl | 2.681 |
| 15 | 0.35 M 0.964 μl | 3.0 |
| 20 | 0.35 M 1.43 μl | 2.54 |
| 30 | 0.35 M 1.93 μl | 2.04 |
| 56 | 0.35 M 3.6 μl | 0.367 |

10 μl samples from each tube were loaded onto a black flat-bottom 96-well plate as follows (the numbers represent the tube labels / K-G concentration in mM):

| 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 | 11 | 12 |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
|  |  |  |  |  |  |  |  |  |  |  |  |
|  | 0 | 4 | 6 | 8 | 10 | 15 | 20 | 30 | 56 |  |  |
|  |  | 4 | 6 | 8 | 10 | 15 | 20 | 30 | 56 |  |  |
|  |  |  |  |  |  |  |  |  |  |  |  |
|  |  |  |  |  |  |  |  |  |  |  |  |
|  |  |  |  |  |  |  |  |  |  |  |  |
|  |  |  |  |  |  |  |  |  |  |  |  |
|  |  |  |  |  |  |  |  |  |  |  |  |

The reaction was incubated at 37℃ for 3 hrs. The fluorescence intensity at 569/593 nm was measured at 1 min intervals.

No signal was detected.

![A graph of fluorescent intensity in cell-free reaction measured from different samples presented as a function of time.](https://static.igem.wiki/teams/4207/wiki/220714-cell-free.png)

**Figure 13.**
mScarlet-I produced in cell-free reaction.

**14.07.2022 Transformations for DH5α competent cells**

Transformations were made using Heat-shock transformation protocol for *E. coli* cells. Competent DH5α cells were made 13.7.

5 µl DNA was added on the cells (B 46 -> 3 µl). 4 transformation rounds (3 at a time altogether 12 transformations)

round 1 DNA: T 7 term, T7 max, T7prom

round 2 DNA: Tr 3, A 95, B 69

round 3 DNA: B 46, A 92, Tr 2

round 4 DNA: B 39, Tr 1, A 70

picture 220714 plates

![A picture of bacteria in plates. All plates show a lot of colonies.](https://static.igem.wiki/teams/4207/wiki/220714-plates.png)

**Figure 14.**
A. T7 max in *E.coli* DH5 α. B. Trigger 3 in *E.coli* DH5 α. C. A95 in *E.coli* DH5 α. D. B69 in *E.coli* DH5 α. E. T7 promoter in *E.coli* DH5 α. F. T7 terminator in *E.coli* DH5 α.

![A picture of bacteria in plates. All plates show a lot of colonies.](https://static.igem.wiki/teams/4207/wiki/220714-plates-2.png)

**Figure 15.**
A. A92 in *E.coli* DH5 α. B. A70 in *E.coli* DH5 α. C. B46 in *E.coli* DH5 α. D. Trigger 1 in *E.coli* DH5 α. E. Trigger 2 in *E.coli* DH5 α. F. B39 in *E.coli* DH5 α.

**Gel electrophoresis for lacZ fragments**

60 ml of 1 x TAE buffer was heated with 0.60 g agarose. 6 µl of SYBR safe color was added and mixed. Left to cool. Poured onto a gel tray. 6 µl of Gel Loading Dye 6X was added to the sample (sample volume 25 µl). 7 µl of 1 kb Quick-Load DNA Plus Ladder Purple was loaded onto the gel. All of the sample was loaded onto the gel. Gel was run at 100 V for 35 mins.

**14.07.2022 ZikaV trigger RNA purification attempt**

RNA was attempted to purify with Monarch total RNA miniprep kit. 2 volumes (20 µl) of RNA lysis buffer was added to the 20 µl reaction and mixed with pipette. 60 µl RNA wash buffer (mistake) was added and mixed with pipette. The mixture was transferred to an RNA purification column and spinned for 30 seconds, the flow-through was discarded. 500 µl of RNA Priming Buffer was added and spinned for 30 seconds. Then the mistake was noticed, so the purification was not continued.

**Zika V trigger RNA synthesis**

Two reactions were made. To the reaction tubes, the reagents were added in the following order: 1.5 µl 10 X RNAP reaction buffer; 1.5 µl each of the NTPs: ATP (100 mM), GTP (100 mM), CTP (100 mM), UTP (100 mM); 1 ug, 11 µl, Zika V trigger DNA (87.24 ng/µl), 1.5 µl T7 RNAP. 20 µl reaction. After final addition, mixed with pipette. The reactions were incubated at 37℃.

**Zika-T RNA purification**

Both ZikaV-T RNA tubes were purified using Monarch total RNA miniprep kit (according to our protocol). Concentrations for tubes (measured with nanodrop) were 160 ng/µl and 423.5 ng/µl. Stored in -20℃.

15.07.2022 RNA gel electrophoresis, overnight cultures of the transformed plasmids

Present: Jonna, Anni, Kristiina, Kevät, Jesper, Sofia

**Golden gate minipreps**

12 Golden gate minipreps (DH5a + T7 term, T7 max, T7prom, Tr 3, A 95, B 69, B 46, A 92, Tr 2, B 39, Tr 1, A 70) were made 5ml + kanamycin, overnight 37℃ in shaker.

**15.07.2022 RNA gel electrophoresis**

Agarose gel was made. 60 ml of 1 x TAE buffer was heated with 0.60 g agarose. 6 µl of SYBR safe color was added and mixed. Left to cool. Loaded and run.

![A picture of agarose gel electrophoresis for ZikaV trigger RNA. Very faint bands shown in the upper part of the gel.](https://static.igem.wiki/teams/4207/wiki/220715-gel.png)

**Figure 16.**
Agarose gel electrophoresis for ZikaV trigger RNA.

16.07.2022 Glycerol preps from the transformed plasmids

Present: Anni, Kevät

**Glycerol preps**

Glycerol preps were made using 875 µl of each sample and 125 µl glycerol.

samples: DH5a + T7 term, T7 max, T7prom, Tr 3, A 95, B 69, B 46, A 92, Tr 2, B 39, Tr 1, A 70

stored in -80℃.

18.07.2022 Human Practices meeting

Present: Jonna, Juuli, Kevät, Malin, Nelli, Otto

We planned our educational material further and shared responsibilities regarding it.

![A picture of the Zoom view.](https://static.igem.wiki/teams/4207/wiki/aboa-notebook-220718hp.png)

18.07.2022 Plasmid extraction, Golden gate assembly of triggers and toeholds, cultures for BL21 Gold-dLac and BL21 pET15b-LacZ

Present: Jonna, Anni, Kevät

**Plasmid minipreps**

Plasmid minipreps were prepared from the cultures stored in the fridge on Saturday, with Monarch Miniprep Kit (according to manufacturer's protocol). In the last step, the elution was done in 21 µl water. Stored in -20℃, in the GoldenGate box.

Concentrations: shown in the image below.

**Golden Gate Assembly of the triggers and the toeholds**

A total of 9 reactions were made. Tubes 1-3 contained pODD1 plasmid, T7 promoter and a trigger, and tubes 4-9 contained pODD1, T7 promoter, a toehold switch, LacZ and terminator, according to the tables below. The concentrations needed are calculated below.

![A table with calculations.](https://static.igem.wiki/teams/4207/wiki/220718-pic.png)

**Figure 17.**
Calculations of needed concentrations for golden gate reactions.

| A | B | C | D |  |  |  |
| --- | --- | --- | --- | --- | --- | --- |
| pODD1 (µl) | T7 promoter | Buffer | Enzyme | H2O |  |  |
| 1 | 0.31 | TR1 | 0.17 | 2 | 1 | 15.52 |
| 1 | 0.31 | TR2 | 0.22 | 2 | 1 | 15.47 |
| 1 | 0.31 | TR3 | 0.27 | 2 | 1 | 15.42 |

| pODD1 (µl) | T7 promoter | Toehold | LacZ | Terminator | Buffer | Enzyme | H2O |
| --- | --- | --- | --- | --- | --- | --- | --- |
| 1 | 0.31 | A92 | 0.29 | 0.36 | 0.32 | 2 | 1 | 14.72 |
| 1 | 0.31 | B69 | 0.27 | 0.36 | 0.32 | 2 | 1 | 14.74 |
| 1 | 0.31 | B46 | 0.31 | 0.36 | 0.32 | 2 | 1 | 14.70 |
| 1 | 0.31 | A95 | 0.24 | 0.36 | 0.32 | 2 | 1 | 14.77 |
| 1 | 0.31 | B39 | 0.30 | 0.36 | 0.32 | 2 | 1 | 14.71 |
| 1 | 0.31 | A70 | 0.29 | 0.36 | 0.32 | 2 | 1 | 14.72 |
|  |  |  |  |  |  |  |  |  |

Golden gate reaction protocol (according to instructions on NEB site): (1 min 37℃, 1 min 16℃) X 30, 5 min 60℃, keep on 4℃ for infinity.

Stored at -20℃.

**Growth cultures BL21 Gold deLac and BL21 pET15 lacZ**

A streak of BL21 Gold deLac freeze stock was inoculated in 5 ml of YT + 5 µl tetracycline (stock concentration 10 mg/ml). A streak of BL21 pET15 lacZ containing plate was inoculated in 5 ml of YT + 5 µl ampicillin (stock concentration 100 mg/ml). Kept overnight in a shaker at 37℃.

19.07.2022 Visual Design meeting

Present: Juuli, Minttu, Nelli, Otto

We filmed the majority of the material for our promotion video.

19.07.2022 Transformation for Golden Gate assembled plasmids, growth culture for BL21 Gold-dLac

Present: Anni, Kristiina, Kevät, Sofia

**Preparing 0,1 M MgCl2**

4.062 g of MgCl2 x H2O was diluted into ~ 100 ml of MQ water. Filled up to 200 ml of MQ water. Autoclaved.

**Growth/starter culture BL21 Gold deLac**

A streak of BL21 Gold deLac freeze stock was inoculated in 5 ml of YT + 5 µl tetracycline (stock concentration 10 mg/ml) same as yesterday. Kept overnight in a shaker at 37℃.

**Transformation for Golden Gate assembled plasmids (3+6)**

Transformations were made using the Heat-shock transformation protocol for *E. coli* cells.

![A picture of bacteria in plates. All plates show colonies.](https://static.igem.wiki/teams/4207/wiki/220719-plates-1.png)

**Figure 18.**
A. Golden Gate assembled pOdd1 T7+A92+LacZ+terminator in E.coli DH5 α. B. Golden Gate assembled pOdd1 T7+A92+LacZ+terminator in E.coli DH5 α. C. Golden Gate assembled pOdd1 T7+A95+LacZ+terminator in E.coli DH5 α. D. Golden Gate assembled pOdd1 T7+A95+LacZ+terminator in E.coli DH5 α. E. Golden Gate assembled pOdd1 T7+A70+LacZ+terminator in E.coli DH5 α. F. Golden Gate assembled pOdd1 T7+A70+LacZ+terminator in E.coli DH5 α.

![A picture of bacteria in plates. All plates show colonies.](https://static.igem.wiki/teams/4207/wiki/220719-plates-2.png)

**Figure 19.**
A. Golden Gate assembled pOdd1 T7+B46+LacZ+terminator in E.coli DH5 α. B. Golden Gate assembled pOdd1 T7+B46+LacZ+terminator in E.coli DH5 α. C. Golden Gate assembled pOdd1 T7+B69+LacZ+terminator in E.coli DH5 α. D. Golden Gate assembled pOdd1 T7+B69+LacZ+terminator in E.coli DH5 α. E. Golden Gate assembled pOdd1 T7+B39+LacZ+terminator in E.coli DH5 α. F. Golden Gate assembled pOdd1 T7+B39+LacZ+terminator in E.coli DH5 α.

![A picture of bacteria in plates. All plates show colonies.](https://static.igem.wiki/teams/4207/wiki/220719-plates-3.png)

**Figure 20.**
A. Golden Gate assembled pOdd1 T7+trigger 3 in E.coli DH5 α. B. Golden Gate assembled pOdd1 T7+trigger 3 in E.coli DH5 α. C. Golden Gate assembled pOdd1 T7+trigger 1 in E.coli DH5 α. D. Golden Gate assembled pOdd1 T7+trigger 1 in E.coli DH5 α. E. Golden Gate assembled pOdd1 T7+trigger 2 in E.coli DH5 α. F. Golden Gate assembled pOdd1 T7+trigger 2 in E.coli DH5 α.

20.07.2022 Team meeting

Present: Evelina, Iida, Jonna, Juuli, Kevät, Kristiina, Malin, Minttu, Nelli

Besides other updates, we discussed recent fundraising activities and possible ways to improve our financial situation. In addition, we planned upcoming events such as Aalto-Helsinki’s visit to Turku and our visit to Linköping for the Nordic iGEM Conference.

![A picture of the Zoom window.](https://static.igem.wiki/teams/4207/wiki/aboa-notebook-meeting28.png)

20.07.2022 Gel electrophoresis for RNA, cultures for ZikaV trigger and BL21 pET15b-LacZcells

Present: Evelina, Anni, Kristiina, Kevät, Sofia

**BL21 Gold deLac Competent cells**

Competent cells were made using protocol for competent cells. 60 ml production culture was made instead of 100 ml (we made half of what the protocol said). When OD600 were 0.4-0.5 the cultures were divided into falcon tubes. 2 falcons, each 25 ml of the culture. Stored at -80℃.

**RNA gel electrophoresis**

Gel electrophoresis for ZikaV Trigger RNA.

Agarose gel was made for the purified Zika V trigger RNA. 50 ml of 1 x TAE buffer was heated with 0.60 g agarose. 5 µl of SYBR safe color was added and mixed. Left to cool.

Sample 1: 18 µl formamide, 5 µl loading dye, 7 µl Zika V trigger RNA (160 ng/µl)

Sample 2: 18 µl formamide, 5 µl loading dye, 7 µl  Zika V trigger RNA (423.5 ng/µl)

sample = 30 µl.

Also 7 µl of 1 kb ladder and 7 µl 100 bp ladder was loaded.

Before loading the samples to the wells, samples were incubated for 50 min at 60°C and left on ice for about 5 min.

100 V was used to run the gel for 10 minutes. Then the voltage was raised by ten after every ten minutes. 110 V after 10 min, 120 V after 20 min, 130 V after 30 min, 140 V after 40 min, 150 V after 50 min and 160 V after 60 min. The gel was run for 70 min.

![A picture of agarose gel electrophoresis. No bands are shown on the gel.](https://static.igem.wiki/teams/4207/wiki/220720-gel.png)

**Figure 21.**
Agarose gel electrophoresis for ZikaV trigger RNA.

Gel run failed again :)

**Zika V trigger overnight miniprep**

5 ml LB, 5 µl amp 100 and Zika V trigger from plate. Grown overnight in shaker 37℃.

**BL21 pET15 lacZ growth culture for the measurement test**

A streak of a plate containing BL21 pET15 lacZ was inoculated in 5 ml of 2 x YT and 5 µl of ampicillin. Grown overnight at ~ 200 rpm at 37°C. Stored in the refrigerator for ~ 5 hours before induction.

21.07.2022 Aalto-Helsinki visiting Turku

![People sitting on the chairs of a cathedral.](https://static.igem.wiki/teams/4207/wiki/aboa-notebook-220721ah.jpg)

21.07.2022 Gel electrophoresis for trigger RNA, plasmid extraction for Zika V trigger, IPTG induction for BL21 pET15b-LacZ

Present: Evelina, Kristiina, Kevät, Jesper

**RNA gel electrophoresis**

Gel electrophoresis for ZikaV Trigger RNA.

Agarose gel was made for the purified Zika V trigger RNA. 50 ml of 1 x TAE buffer was heated with 0.60 g agarose. 5 µl of SYBR safe color was added and mixed. Left to cool.

Sample 1: 18 µl formamide, 5 µl loading dye, 7 µl Zika V trigger RNA (160 ng/µl)

Sample 2: 18 µl formamide, 5 µl loading dye, 7 µl Zika V trigger RNA (423.5 ng/µl)

sample = 30 µl.

Also 7 µl of 1 kb ladder and 7 µl 100 bp ladder was loaded.

Before loading the samples to the wells samples were incubated for 50 min at 60°C and left on ice for about 5 min.

100 V was used to run the gel for 10 minutes. Then the voltage was raised by ten volts after every ten minutes. 110 V after 10 min, 120 V after 20 min, 130 V after 30 min, 140 V after 40 min, 150 V after 50 min. The gel was run for 60 min.

![A picture of agarose gel electrophoresis. Very faint bands shown in the upper part of the gel.](https://static.igem.wiki/teams/4207/wiki/220721-gel.png)

**Figure 22.**
Agarose gel electrophoresis for ZikaV trigger RNA.

Gel run failed.

**Plasmid extraction Zika V trigger**

Plasmids were purified using the NEB plasmid miniprep kit. The first centrifuge step was done as follows: 5 mins, 3000 rcf. The plasmids were elued to 50 µl elution buffer.

The concentrations of the DNA in the samples were determined by using BioDrop. Concentration 117.0 ng/µl, A260/A230=1.158, A260/A280=1.857. Stored in the freezer.

**IPTG induction for BL21 pET15 lacZ**

100 µl of BL21 pET15 lacZ cell culture was inoculated in 10 ml of 2 x YT + 10 µl ampicillin in Erlenmeyer. Grown until OD600 was about 0.52. The total volume of the culture was 7 ml after taking 3 ml for measuring OD. 0.35 µl of IPTG (stock concentration 0.2 M) was added into the culture. IPTG concentration was 1 mM in the culture. Grown at room temperature overnight.

22.07.2022 Transformation for pEAS002 (mScarlet-I), test with IPTG induced cells, ZikaV trigger plasmid linearization, purification and RNA synthesis

Present: Evelina, Anni, Kristiina, Kevät, Jesper, Sofia

**Measurement test with IPTG induced cells**

7 ml of *E. coli* BL21 DE3 pET15b-LacZ cells from overnight induction were supplemented with 63.25 μl of 10 mg/ml ONPG in DMSO.

10, 20, 40 and 60 μl samples were loaded onto a clear, V-bottom 96-well plate as follows:

| 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 | 11 | 12 |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
|  |  |  |  |  |  |  |  |  |  |  |  |
|  |  |  |  |  |  |  |  |  |  |  |  |
|  |  |  |  |  |  |  |  |  |  |  |  |
|  | 10 | 10 | 20 | 20 | 40 | 40 | 60 | 60 |  |  |  |
|  | 10 | 10 | 20 | 20 | 40 | 40 | 60 | 60 |  |  |  |
|  |  |  |  |  |  |  |  |  |  |  |  |
|  |  |  |  |  |  |  |  |  |  |  |  |
|  |  |  |  |  |  |  |  |  |  |  |  |

Reactions were incubated without agitation at 29℃ for 3 hrs and absorbance at 420 nm was measured at 1 min intervals.

![A graph of absorbance measured in cell-free reaction as a function of time with different samples.](https://static.igem.wiki/teams/4207/wiki/220722-cell-free.png)

**Figure 23.**
β-galactosidase activity in cell-free reaction.

**22.07.2022 Transformation for BL21-Gold-dLac comp.**

Transformation for BL21-Gold-dLac comp. and mScarlet-I (pEAS002) were made using the Heat-shock transformation protocol for *E. coli* cells.

The transformation was made using 50 µl of competent BL21-Gold-dLac cells. 10 µl DNA was added to the cells.

**Zika V trigger plasmid linearisation**

4 50 µl reactions were made. To the tubes were pipetted the following reagents: 35.4 µl autoclaved MQ-water, 8.6 µl Zika V trigger plasmid DNA (1 µg, 117.0 ng/µl), 5 µl NEBuffer 3, 1 µl Pst1 restriction enzyme. The reactions were incubated at 37℃ for 15 min.

**Zika V trigger DNA purification**

Linearised plasmid were purified with NEB Monarch PCR & DNA cleanup kit, according to the kit instructions. The elution was made to 12 µl elution buffer. Concentrations and purity were measured with BioDrop:

| sample | c (ng/µl) | A260/A230 | A260/A280 |
| --- | --- | --- | --- |
| 1 | 27.18 | 1.582 | 1.790 |
| 2 | 31.85 | 1.784 | 1.890 |
| 3 | 36.10 | 1.890 | 1.890 |
| 4 | 32.87 | 1.742 | 1.839 |

**Zika V trigger RNA synthesis**

2 RNA synthesis reactions were made. Samples 1 and 2 were combined (20 µl, total 590, 3 ng DNA) and samples 3 and 4 were combined (20 µl, total 689.7 ng DNA). The reagents were pipetted in the tubes in following order: 2 µl of RNAP reaction buffer; 2 µl of each of the NTPs; 20 µl of linearised DNA; 2 µl of T7 RNAP. (32 µl total) The reactions were incubated at 37℃ for 8 hours. The tubes were stored at -20℃.

23.07.2022 Wiki writing session

25.07.2022 ZikaV trigger RNA purification and gel electrophoresis, transformation for BL21-Gold-dLac competent cells, starter cultures

Present: Evelina, Anni, Kristiina, Kevät, Jesper

**O/N cultures of Toeholds A92, A95, A70, B39, B46, B69 and Triggers 1, 2, 3 and pOdd1**

5 ml LB overnight cultures of Toeholds A92, A95, A70, B39, B46, B69 and Triggers 1, 2, 3 were made with KAN50. Two 5 ml LB O/N cultures of pOdd1 were made with KAN50.

**Transformation for BL21-Gold-dLac comp.**

Transformation for BL21-Gold-dLac comp. and mScarlet-I (pEAS002) were made again using the Heat-shock transformation protocol for *E. coli* cells.

The transformation was made using 50 µl of competent BL21-Gold-dLac cells. 10 µl DNA was added to the cells. Cells were plated on a double antibiotic (kanamycin and tetracycline) plate.

Linear plasmid was used by accident and the tubes were discarded.

**Starter culture for LacZ purification**

Single colony from the LBA plate containing BL21 Gold-dLac pET15b-LacZ was inoculated into 5 ml LB + 100 μg/ml ampicillin in a culture tube. Culture was incubated in 37℃, 220 rpm for 16 hours.

**25.07.2022 Zika-T RNA purification**

Both ZikaV-T RNA tubes were purified using Monarch total RNA miniprep kit (according to our protocol). Concentrations for tubes (measured with nanodrop) were 209.7 ng/µl and 257.9 ng/µl. Stored in -20℃.

**RNA gel electrophoresis**

Gel electrophoresis for ZikaV Trigger RNA.

Agarose gel was made for the purified Zika V trigger RNA. 50 ml of 1 x TAE buffer was heated with 0,60 g agarose. 5 µl of SYBR safe color was added and mixed. Left to cool.

Sample 1: 18 µl formamide, 5 µl loading dye, 7 µl Zika V trigger RNA (160 ng/µl)

Sample 2: 18 µl formamide, 5 µl loading dye, 7 µl Zika V trigger RNA (423.5 ng/µl)

sample = 30 µl.

Also 7 µl of 1 kb ladder and 7 µl 100 bp ladder was loaded.

Before loading the samples to the wells, samples were incubated for 50 min at 60°C and left on ice for about 5 min.

100 V was used to run the gel for 20 minutes. Then the voltage was raised to 120 V for 30 min.

![A picture of agarose gel electrophoresis. No bands on the sample wells can be seen.](https://static.igem.wiki/teams/4207/wiki/220725-gel.png)

**Figure 24.**
Agarose gel electrophoresis for ZikaV trigger RNA.

26.07.2022 Cell-free reaction with pET15b-LacZ plasmid, transformation for BL21-Gold-dLac comp., plasmid extractions, Tris buffer and phosphate buffer preparation and ONPG dilution

Present: Anni, Kristiina, Kevät, Jesper, Sofia

**Cell-free reaction with pET15b-LacZ**

Using the master mix calculator with 230 μl as the final volume, the following mastermix was created into tube 1 (positive sample):

| Reagent | Volume (μl) |
| --- | --- |
| Water | 40.57 |
| Amino acid mix | 46 |
| HMP | 4.6 |
| K-Glut | 3.68 |
| Mg-Glut | 3.4 |
| Energy mix | 23 |
| PEG | 11.5 |
| IPTG | 1.15 |
| ONPG | 2.07 |
| Extract | 84.14 |

28.71 μl of the master mix was transferred to tube 2 along with 1.28 μl of water.

8.58 μl of pET15b-LacZ DNA (62.66 ng/μl) was added to tube 1.

A 30 μl sample from tube 2 (water control) and 2 x 20 μl, 2 x 30 μl and 2 x 40 μl samples from tube 1 were transferred to a clear v-bottom 96 well-plate as follows:

| 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 | 11 | 12 |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
|  |  |  |  |  |  |  |  |  |  |  |  |
|  | Water | 20 | 30 | 40 |  |  |  |  |  |  |  |
|  |  | 20 | 30 | 40 |  |  |  |  |  |  |  |
|  |  |  |  |  |  |  |  |  |  |  |  |
|  |  |  |  |  |  |  |  |  |  |  |  |
|  |  |  |  |  |  |  |  |  |  |  |  |
|  |  |  |  |  |  |  |  |  |  |  |  |
|  |  |  |  |  |  |  |  |  |  |  |  |

The reactions were incubated at 29℃ for 3 hrs and absorbance at 420 nm was measured at 1 min intervals.

![A graph of absorbance measured in cell-free reaction as a function of time with different samples.](https://static.igem.wiki/teams/4207/wiki/220726-cell-free.png)

**Figure 25.**
β-galactosidase activity in cell-free reaction.

**Glycerol preps**

Glycerol preps were made using 875 µl of each sample and 125 µl glycerol.

samples: DH5α + pOdd1 + Tr 1 or Tr 2 or Tr 3, or A 95 or B 69 or B 46 or A 92 or B 39 or A 70

--> stored in -80℃

**Plasmid extractions Toeholds A92, A95, A70, B39, B46, B69 and Triggers 1, 2, 3**

Extraction was made using the NEB Monarch Plasmid Miniprep Kit, according to the manufacturer's protocol. First spin was done in 3000 x g 5 min. Concentrations measured with BioDrop:

| Sample | Concentration ng/µl | A260/280 | A260/A230 |
| --- | --- | --- | --- |
| A92 | 166.7 | 1.880 | 1.992 |
| A95 | 141.7 | 1.872 | 1.976 |
| A70 | 132.9 | 1.874 | 1.874 |
| B39 | 133.4 | 1.895 | 1.895 |
| B46 | 135.9 | 1.864 | 1.864 |
| B69 | 141.5 | 1.899 | 1.952 |
| T1 | 27.71 | 1.884 | 0.847 |
| T2 | 31.79 | 2.014 | 1.032 |
| T3 | 26.70 |  |  |

**Transformation for BL21-Gold-dLac comp**

THIRD TIME :) Transformation for BL21-Gold-dLac comp. and mScarlet-I (pEAS002) were made again using the Heat-shock transformation protocol for *E. coli* cells.

The transformations were made using 50 µl of competent BL21-Gold-dLac cells. 10 µl DNA was added to the cells.

--> Plated on a double antibiotic (kanamycin and tetracycline) plate.

**Tris A buffer, 200ml, pH 7.5**

20 mM Tris-HCl = 4 ml 1M Tris-HCl stock

150 mM NaCl = 1.753 g

20 mM imidazole = 0.272 g

to 200 ml MQ H2O

pH was adjusted to 7.5 with HCl. Stored in 4℃.

**Phosphate buffer 0.08 M 100 ml, pH 7,7**

KH2PO4 0.369 g

K2HPO4 0.921 g

pH was adjusted to 7.7 with KOH. Stored in 4℃.

**10 mg/ml ONPG in DMSO**

10.6 mg ONPG

1060 ml DMSO

Stored in -20℃.

**ONPG in PBS**

10.58 µl 10 mg/ml ONPG in DMSO

89.42 µl 1X PBS

**O/N cultures of Trigger 1, 2, 3**

5 ml overnight cultures of DH5ɑ pOdd1 Trigger 1, 2, 3 in LB with KAN50.

**Production culture for LacZ purification**

4 ml of overnight culture was added to 200 ml of 2xYT with about 70 μg/ml ampicillin. When OD600 reached 0.613, 1 ml of 0.2 M IPTG was added and incubation was continued at 220 rpm, 23℃ for 3 hrs.

27.07.2022 Team meeting

Present: Anni, Jesper, Juuli, Malin, Nelli, Otto, Sofia

We updated each other about recent things as well as discussed our collaborations, Grand Jamboree, and Integrated Human Practices activities.

![A picture of the Zoom window.](https://static.igem.wiki/teams/4207/wiki/aboa-notebook-meeting29.png)

27.07.2022 Sonication and purification of LacZ

Pellet suspension was thawed on ice and then sonicated for 4x30 s. Suspension was centrifuged at 48 000 g, 4℃ for 30 min.

Protein was purified according to the following protocol:

1 ml of Ni2+ sepharose fast flow was pipetted into a filter tube

Ethanol was washed away with 4 ml of water

The column was balanced with 4 ml of Tris A buffer

The supernatant was filtered through column

Column was washed with 4 ml of Tris A buffer

5 fractions were collected, each with 1 ml of Tris B buffer pipetted into the column

FInally the Ni2+ was collected with 10 ml of water

Purification was unsuccessful, as we got very little protein.

28.07.2022 Meeting with Patras and TecCEM

28.07.2022 Restriction analysis of toehold plasmids and linearisation of trigger plasmids, purification and RNA synthesis of linearised trigger plasmids

Present: Anni, Jesper, Sofia

**Restriction analysis of toehold plasmids & linearisation of trigger plasmids**

51 µl reactions digestion reactions for each of the toehold (EcoRI-HF) and trigger (PstI-HF) plasmids were composed of the following reagents:

| Plasmid | Volume to get 1 μg of plasmid (µl) | rCutSmart Buffer (µl) | Restriction enzyme (µl) | Water (µl) |
| --- | --- | --- | --- | --- |
| A70 | 7.5 | 5 | 1 | 37.5 |
| A92 | 6 | 5 | 1 | 39 |
| A95 | 7.1 | 5 | 1 | 37.1 |
| B39 | 7.5 | 5 | 1 | 37.5 |
| B46 | 7.4 | 5 | 1 | 37.6 |
| B69 | 7.1 | 5 | 1 | 37.9 |
| Trigger 1 | 7.7 | 5 | 1 | 37.3 |
| Trigger 2 | 4.5 | 5 | 1 | 40.5 |
| Trigger 3 | 4.8 | 5 | 1 | 40.2 |

Normally the reaction volume is not 51 µl, but the volume of the enzyme was forgotten about the calculations.

The reaction was incubated was 37℃ for 4 hours, followed by 20 minutes at 80℃.

**Purification of linearised toehold and trigger plasmids.**

Purifications were made using Monarch PCR & DNA Cleanup Kit.

After purification the triggers DNA concentrations were measured with BioDrop:

Trigger 1: 0.125 μg/ul

Trigger 2: 0.105 μg/ul

Trigger 3: 0.103 μg/ul

After measure there were left 5 µl each trigger sample.

**RNA synthesis of triggers**

RNA synthesis of triggers 1, 2 and 3 were made using the RNA production protocol.

Because the concentration of the each DNA were too low, all of the DNA were used in the synthesis reaction and the reaction volumes were reduced to 15 µl.

Was left to incubate at 37℃ overnight.

29.07.2022 Restriction analysis of toehold plasmids, RNA purification

Present: Anni, Jesper, Sofia

**Gel electrophoresis for toehold plasmids**

10 µl of 6X Purple loading dye was added to digested 51 µl samples. 20 µl of these samples were loaded to the gel, and electrophoresis was run at 100 V for 40 minutes.

Samples were pipetted to the gel in the following order:

| Ladder | Ladder | A70 | A92 | A95 | Ladder | B39 | B46 | B69 |
| --- | --- | --- | --- | --- | --- | --- | --- | --- |

![A picture of agarose gel electrophoresis. Multiple bands with a lot of background can be seen in all the sample wells.](https://static.igem.wiki/teams/4207/wiki/220729-gel.png)

**Figure 26.**
Agarose gel electrophoresis for digested plasmids.

There should be two bands, 2.4 kb and 3.2 kb. Those bands cannot be seen on the gel, the plasmid is most likely undigested.

**Restriction analysis of toehold plasmids again**

Since last time the plasmids didn't digest properly, this time more enzyme is added

20 µl reactions as follows:

| Plasmid | Volume to get 1 μg of plasmid (µl) | rCutSmart Buffer (µl) | Restriction enzyme (µl) | Water (µl) |
| --- | --- | --- | --- | --- |
| A70 | 7.5 | 2 | 1 | 9.5 |
| A92 | 6 | 2 | 1 | 11 |
| A95 | 7.1 | 2 | 1 | 9.9 |
| B39 | 7.5 | 2 | 1 | 9.5 |
| B46 | 7.4 | 2 | 1 | 9.6 |
| B69 | 7.1 | 2 | 1 | 9.9 |
| pos. control | 15 | 2 | 1 | 2 |
| neg. control | 6 | 2 | 0 | 12 |

pos control: pET-15b lacZ

Negative control: A92 toehold, no enzyme.

Incubation in 37℃

![A picture of agarose gel electrophoresis. Multiple bands can be seen on the sample wells.](https://static.igem.wiki/teams/4207/wiki/220729-gel-2.png)

**Figure 27.**
Agarose gel electrophoresis for digested plasmids

**RNA purification of the triggers 1, 2 and 3**

RNA purification to the triggers were made using RNA production protocol.

After purification the concentrations of the triggers RNA were measured with biodrop:

Trigger 1: 416.1 ng/μl

Trigger 2: 673.5 ng/μl

Trigger 3: 385.6 ng/μl

Stored in the freezer (-20℃).

31.07.2022 Starter cultures for protein purification

Present: Jesper

A single colony of BL21 pET15b-LacZ was inoculated into a culture tube containing 5 ml of 2xYT + ampicillin at 100 μg/ml.

A single colony of BL21 Gold-dLac pEAS002 was inoculated into a culture tube containing 5 ml of 2xYT + tetracycline at 10 μg/ml and kanamycin at 50 μg/ml.

Cultures were incubated at 37°C, 220 rpm for 12 hrs.

AUG

01.08.2022 Cell-free reaction with mScarlet-I, production cultures for protein purification

Present: Kevät, Jesper

**Cell-free reaction test with mScarlet-I**

This time, we have a couple of modifications. We changed the amount of cell extract so the final concentration is 10 mg/ml (before was 15 mg/ml) of protein. We also changed the plate (black PCR plate with clear wells) and we are testing bigger concentrations, as we did last week.

Mastermix amounts were calculated using 230 μl as the total volume. The following amounts were to tube labeled 1 (positive sample master mix). Each stock tube was and the mastermix were vortexed before each addition until cell extract, which we did not vortex. Pipetted as follows:

| Reagent | Volume (μl) |
| --- | --- |
| Water | 74.87 |
| Amino acid mix | 46 |
| HMP | 4.6 |
| K-Glut | 3.68 |
| Mg-Glut | 3.4 |
| Energy mix | 23 |
| PEG | 11.5 |
| IPTG | 2.07 |
| Extract | 56.7 |

29.26 μl of the mix was transferred to a tube labelled 2 (water control). 0.74 μl of water was added to it.

4.95 μl of DNA was added to tube 1.

A single 30 μl water control sample was pipetted onto the plate. 2 x 20 μl, 2 x 30 μl and 2 x 40 μl samples were pipetted onto the plate and reactions were incubated at 29℃ for 3 hrs. Fluorescence at 569/593 nm was measured at 1 min intervals.

![A graph of fluorescent intensity in cell-free reaction measured from different samples presented as a function of time.](https://static.igem.wiki/teams/4207/wiki/220801-cell-free.png)

**Figure 28.**
mScarlet-I produced in cell-free reaction.

**Production cultures for protein purification**

Overnight cultures were each added to 400 ml of 2xYT with their appropriate antibiotics. Incubation at 37°C, 220 until cells reached OD600 0.6. 2 ml of 0.2 M IPTG was added and incubation started at 220 rpm, 23°C.

pEAS002 reached 0.059 OD600 at 12.55. IPTG was added as above.

02.08.2022 Trigger plasmid linearization, purification and gel electrophoresis, RNA synthesis, protein purification for mScarlet-I (pEAS002) and β-galactosidase, 2 M imidazole and Tris B buffer preparation, LBA plates

Present: Evelina, Kristiina, Kevät, Jesper

**10 x LBA+KAN50 plates**

Approximately 200 ml of melted LBA was put into a flask. 200 µl of kanamycin (stock concentration 50 mg/ml) was added. Mixed. Poured onto 10 plates.

**Trigger plasmid linearization**

50 µl reactions were made to all 3 triggers. To the tubes were pipetted the following reagents: 35.4 µl autoclaved MQ-water, 1 µg trigger plasmid DNA, 5 µl buffer, 1 µl Pst1 restriction enzyme. The reactions were incubated at 37°C for 2 h and 80°C for 20 min.

**Linearized trigger plasmid purification**

Linearized plasmids were purified with NEB Monarch PCR & DNA cleanup kit, according to the kit instructions. The elution was made to 11.5 µl elution buffer. Concentrations and purity were measured with BioDrop:

| sample | c (ng/µl) | A260/A230 | A260/A280 |
| --- | --- | --- | --- |
| 1 | 57.69 | 1.943 | 1.880 |
| 2 | 51.41 | 1.875 | 1.875 |
| 3 | 49.35 | 1.947 | 1.873 |

**Trigger RNA synthesis**

RNA synthesis reactions were made to all 3 samples. The reagents were pipetted in the tubes in following order: 1.5 µl of RNAP reaction buffer; 1.5 µl of each of the NTPs; 5 µl of linearised DNA; 1.5 µl of T7 RNAP. (14 µl total) The reactions were incubated at 37℃ overnight.

**Gel electrophoresis for linearized trigger plasmids**

60 ml of 1 x TAE buffer was heated with 0.60 g agarose. 6 µl of SYBR safe color was added and mixed. Left to cool. Poured onto gel tray. Approximately 1 µl of Gel Loading Dye 6X was added to the sample (sample volume 5 µl). 7 µl of 1 kb Quick-Load DNA Plus Ladder Purple was loaded onto the gel. Approximately 6 µl (all of the sample) was loaded onto the gel. Unfortunately, Trigger Plasmid 2 vanished from the well. Gel was run at 100 V for 35 mins.

![A picture of agarose gel electrophoresis. Multiple bands can be seen on sample wells 2 and 4, 2 bands with smudges can be seen on sample well 3.](https://static.igem.wiki/teams/4207/wiki/220802-gel.png)

**Figure 29.**
Agarose gel electrophoresis for ZikaV trigger, triggers 1, 2 and 3 RNA. Wells: 1 kb DNA Plus Ladder, Trigger Plasmid 1, Trigger Plasmid 2, Trigger Plasmid 3.

**Protein purification for mScarlet-I (pEAS002) and β-galactosidase**

Both cell suspensions were thawed on ice and sonicated at peak value of 12 for 4 x 30 s, with 30 s intervals. The sonicated liquids were then centrifuged at 48 434 G, 4°C for 3 minutes. The supernatant was collected and the pellet discarded.

Both proteins were purified using the following protocol:

1 ml of Ni2+ sepharose fast flow was pipetted into a filter tube

Ethanol was washed away with 4 ml of water

The column was balanced with 4 ml of Tris A buffer

The supernatant was filtered through column

Column was washed with 4 ml of Tris A buffer

5 fractions were collected, each with 1 ml of Tris B buffer pipetted into the column

FInally the Ni2+ was collected with 10 ml of water

From each of the samples, the first two fractions were combined (to a volume of 2 ml). They were mixed with 3.2 ml of 80 % glycerol and stored at -20°C for later use.

The fractions did not seem to contain any protein based on a modified Bradford assay, but the first two mScarlet-I fractions clearly contained protein, so we combined the first two from each sample.

**ZikaV Trigger Growth Cultures**

A streak of a plate containing E. coli ZikaV Trigger was inoculated in 5 ml of 2 x YT and 5 µl of ampicillin. Two growth cultures were prepared. Grown overnight at 37°C at 200 rpm in Falcon tubes.

**2 M Imidazole 5 ml**

0.6808 g imidazole (M= 68.08 g/mol)

to 5 ml MQ H2O

**Tris B buffer, 15 ml**

12.75 ml Tris A

2.25 ml 2 M imidazole (total conc. 300 mM)

03.08.2022 Team meeting + Meeting with Uppsala

Present: Iida, Jesper, Jonna, Juuli, Kevät, Kristiina, Nelli

In this week’s team meeting, we discussed the starting of crowdfunding, fundraising otherwise, and wiki-related issues, such as referencing style, responsibilities, and internal deadlines.

![A picture of the Zoom window.](https://static.igem.wiki/teams/4207/wiki/aboa-notebook-meeting30.png)

03.08.2022 Golden Gate assembly for T7 + mScarlet-I + toeholds, trigger RNA purification and gel electrophoresis, linearisation for ZikaV trigger

Present: Evelina, Jonna, Kevät, Jesper

**Golden gate assembly for T7 + mScarlet-I**

6 reactions were made, one for each toehold. All the concentrations were as before (18.7.), but the concentration of the reporter PCR product is 44.77 ng/µl. The components for each of the reactions are:

| pODD1 (µl) | T7 promoter | Toehold | mScarlet-I | Terminator | Buffer | Enzyme | H2O |
| --- | --- | --- | --- | --- | --- | --- | --- |
| 1 | 0.31 | A92 | 0.29 | 0.7 | 0.32 | 2 | 1 | 14.38 |
| 1 | 0.31 | B69 | 0.27 | 0.7 | 0.32 | 2 | 1 | 14.4 |
| 1 | 0.31 | B46 | 0.31 | 0.7 | 0.32 | 2 | 1 | 14.36 |
| 1 | 0.31 | A95 | 0.24 | 0.7 | 0.32 | 2 | 1 | 14.43 |
| 1 | 0.31 | B39 | 0.30 | 0.7 | 0.32 | 2 | 1 | 14.37 |
| 1 | 0.31 | A70 | 0.29 | 0.7 | 0.32 | 2 | 1 | 14.38 |
|  |  |  |  |  |  |  |  |  |

Golden gate reaction protocol (according to instructions on NEB site): (1 min 37℃, 1 min 16℃) X 30, 5 min 60℃, keep on 4℃ for ∞. Stored at -20℃.

**ZikaV Trigger Growth Cultures**

A streak of a plate containing E. coli ZikaV Trigger was inoculated in 5 ml of 2 x YT and 5 µl of ampicillin. Two growth cultures were prepared. Grown overnight at 37°C at 200 rpm in Falcon tubes.

**Trigger RNA purification**

Trigger RNA tubes were purified using Monarch total RNA miniprep kit (according to our protocol). Concentrations for tubes (measured with nanodrop):

| sample | c (ng/µl) | A260/A230 | A260/A280 |
| --- | --- | --- | --- |
| 1 | 311.4 | 1.736 | 1.953 |
| 2 | 913.8 | 2.655 | 1.809 |
| 3 | 806.2 | 2.794 | 2.037 |

**Agarose gel electrophoresis for trigger RNAs**

0.72 g of agarose was dissolved in 60 ml of 1 x TAE + 25 µl of ethidium bromide. Left to solidify. 15 µl of glyoxylate mix was added to 7.5 µl sample and denatured at 60℃ for 50 min. 2.5 µl of DNA Loading Dye and was added just before loading the gel. Run at 100 V for 1h 15 min.

Wells:

1 kb DNA Ladder

Trigger 1 29.7.

Trigger 2 29.7.

Trigger 3. 29.7.

Trigger 1 3.8.

Trigger 2 3.8.

Trigger 3 3.8.

ZikaV Trigger

=> RNAs were once again invisible on the gel. Juha proposed that we could have a meeting in which we go through the whole protocol and talk generally about how to handle RNAs. We also discussed the possibility of using RNA specific stains/dyes instead of SYBR Safe and ethidium bromide.

**ZikaV Trigger Plasmid Linearization**

17.6 µl of ZikaV Trigger Plasmid (c = 56 smth ng/µl), 24.6 µl of MQ Water, 5 µl of rCutSmart and 1 µl of AvrII was added into a PCR tube. Incubated for two hours at 37°C and then for 20 minutes at 80°C.

04.08.2022 Cell-free reaction with mScarlet-I, transformation for Golden Gate assembled plasmids, linearised ZikaV trigger purification and gel electrophoresis, RNA synthesis for ZikaV trigger, LBA plates

Present: Evelina, Kevät, Jesper

**Cell-free reaction with mScarlet-I**

This reaction was meant to be done with LacZ, but we made a mistake and used mScarlet-I instead so we changed the measurement to fluorescence.

The reactions were performed on a black 384-well PCR plate with transparent wells.

We used sonication 2/41 and autolysis batches. The following mastermixes were made into tube labeled 1 (sample):

| Reagent | Volume -sonication batch (μl) | Volume - autolysis batch (μl) |
| --- | --- | --- |
| Water | 20.23 | 13.08 |
| Amino acid mix | 16 | 16 |
| HMP | 1.6 | 1.6 |
| K-Glut | 1.28 | 1.28 |
| Mg-Glut | 1.18 | 1.18 |
| Energy mix | 8 | 8 |
| PEG | 4 | 4 |
| IPTG | 0.4 | 0.4 |
| ONPG | 0.72 | 0.72 |
| Extract | 19.51 | 26.07 |

From both mastermixes, 10.94 μl were transferred into tubes labeled 2 (water control) along with 1.06 μl of water.

6 μl of pEAS002 solution was added to both samples.

From each of the water controls, 10 μl was loaded into the plate. From both of the sample tubes, 2 x 10 μl and 2 x 20 μl reactions were loaded onto the plate. The reactions were incubated at 29°C for 3 hrs, with fluorescence at 569/593 nm measured at 1 min intervals.

![A graph of fluorescent intensity in cell-free reaction measured from different samples presented as a function of time.](https://static.igem.wiki/teams/4207/wiki/220804-cell-free.png)

**Figure 30.**
mScarlet-I produced in cell-free reaction.

**Heat-shock transformation for A70+MS, A92+MS, A95+MS and pEAS002**

Four preps of competent DH5a cells were thawed on ice for 15 min. 5 µl of A70MS, A92+MS, A95+MS and pEAS002 were added to separate tubes. They were incubated on ice for 30 min. Cells were heat-shocked in a 42℃ water bath for 45 s and kept on ice for 7 min. Then, 500 µl of LB was added to each of the tubes and they were placed on 37℃, 200 rpm for 1 hr. From each of the tubes, 50 µl was plated on a LBA + Kan 50 plate and 300 µl on a second plate. They were incubated at 37℃ overnight.

**3 x LBA+TET plates**

51 ml of melted LBA was added into a flask. 51 µl of tetracycline (stock concentration 10 mg/ml) was added. Mixed. Poured onto three plates.

**Transfer into a new plate - lacZ deficient plasmid containing *E. coli***

Bacteria was streaked from the old plate containing lacZ deficient plasmid containing *E. coli* into a new one (LBA+TET10). Grown overnight at 37°C.

**Linearized trigger plasmid purification**

Linearized plasmid were purified with NEB Monarch PCR & DNA cleanup kit, according to the kit instructions. The elution was made to 11 µl elution buffer. Concentrations and purity were measured with BioDrop:

| sample | c (ng/µl) | A260/A230 | A260/A280 |
| --- | --- | --- | --- |
| 1 | 29.84 | 1.884 | 1.884 |
| 2 | 26.72 | 1.700 | 1.815 |

**Gel electrophoresis for linearized trigger plasmids**

60 ml of 1 x TAE buffer was heated with 0.60 g agarose. 6 µl of SYBR safe color was added and mixed. Left to cool. Poured onto gel tray. Approximately 1 µl of Gel Loading Dye 6X was added to the sample (sample volume 5 µl). 7 µl of 1 kb Quick-Load DNA Plus Ladder Purple was loaded onto the gel. Approximately 6 µl (all of the sample) was loaded onto the gel. Gel was run at 100 V for 35 mins.

![A picture of agarose gel electrophoresis. Both sample wells show two faint bands.](https://static.igem.wiki/teams/4207/wiki/220804-gel.png)

**Figure 31.**
Agarose gel electrophoresis for linearised ZikaV triggers. Wells: 1: 1 kb DNA ladder, 2: Zika V trigger plasmid sample 1, 3: Zika V trigger plasmid sample 2.

**Zika V trigger RNA synthesis**

Two RNA synthesis reactions were made. The reagents were pipetted in the tubes in following order: 6 µl H2O; 1.5 µl of RNAP reaction buffer; 1.5 µl of each of the NTPs; 5 µl of linearised DNA; 1.5 µl of T7 RNAP. (20 µl total) The reactions were incubated at 37℃ overnight.

05.08.2022 Cell-free reactions for A92 and B39 toeholds, ZikaV trigger RNA purification

Present: Evelina, Kevät

**Cell-free reactions for A92 and B39 toeholds**

The following reagents were pipetted to tube 2 (A92 pos):

| AA mix | 28.8 µl |
| --- | --- |
| HMP | 2.88 µl |
| K-G | 2.304 µl |
| Mg-G | 2.1312 µl |
| Energy mix | 14.4 µl |
| PEG | 7.2 µl |
| ONPG | 1.301 µl |
| Extract | 35.12 µl |

7.8 µl was transferred to tube 1 (water control) with 4.2 µl water.

28.76 µl was trasferred to tubes 4 (B39 pos) and 6 (ZikaV pos).

0.57 µl A92 was added to tube 2 and 14.67 µl transferred to tube 3 (A92 neg) with 7.33 µl water. 0.3 µl trigger 1 RNA and 7.04 µl water was added to tube 2.

0.61 µl B39 was added to tube 4 and 14.69 µl transferred to tube 5 (B39 neg) with 7.32 µl water. 0.3 µl trigger 1 RNA and 7.02 µl water was added to tube 4.

0.31 µl Zika sensor was added to tube 6 and 14.54 µl transferred to tube 7 (ZikaV neg) with 7.46 µl water. 0.32 µl trigger RNA and 7.14 µl water was added to tube 6.

10 µl samples were pipetted to 384 plate:

| 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 | 11 | 12 | 13 | 14 | 15 | 16 | 17 | 18 | 19 | 20 | 21 | 22 | 23 | 24 |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
|  |  |  |  |  | 1 | 2 | 2 | 4 | 4 |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
|  |  |  |  |  |  | 3 | 3 | 5 | 5 |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
|  |  |  |  |  |  | 6 | 6 | 7 | 7 |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |

The reactions were incubated at 29°C for 3 hrs, with fluorescence at 569/593 nm measured at 1 min intervals.

![A graph of fluorescent intensity in cell-free reaction measured from different samples presented as a function of time.](https://static.igem.wiki/teams/4207/wiki/220805-cell-free.png)

**Figure 32.**
β-galactosidase activity in cell-free reaction.

**Zika V trigger RNA purification**

Trigger RNA tubes were purified using Monarch total RNA miniprep kit (according to our protocol). Concentrations for tubes (measured with nanodrop):

| sample | c (ng/µl) | A260/A230 | A260/A280 |
| --- | --- | --- | --- |
| 1 | 312.5 | 4.448 | 1.829 |
| 2 | 280.8 | 2.507 | 1.857 |

05.-07.08.2022 Nordic iGEM Conference

![People standing and holding a prize.](https://static.igem.wiki/teams/4207/wiki/aboa-notebook-nic.jpg)

08.08.2022 LBA plates

Present: Evelina, Kristiina, Jesper

![A graph of fluorescent intensity in cell-free reaction measured from different samples presented as a function of time.](https://static.igem.wiki/teams/4207/wiki/220808-cell-free.png)

**Figure 33.**
β-galactosidase activity in cell-free reaction.

15 LBA+KAN50 plates were made and stored in the fridge (+4°C).

09.08.2022 Meeting with Aalto-Helsinki

09.08.2022 Purification of linearized triggers, IPTG preparation

Present: Evelina, Kristiina, Jesper

![A graph of fluorescent intensity in cell-free reaction measured from different samples presented as a function of time.](https://static.igem.wiki/teams/4207/wiki/220809-cell-free.png)

**Figure 34.**
β-galactosidase activity in cell-free reaction.

**Linearized trigger plasmid purification**

Linearised plasmid were purified with NEB Monarch PCR & DNA cleanup kit, according to the kit instructions. The elution was made to 10 µl elution buffer. Concentrations and purity were measured with BioDrop:

| sample | c (ng/µl) | A260/A230 | A260/A280 |
| --- | --- | --- | --- |
| Zika trigger | 33.05 | 1.319 | 1.831 |
| Zika trigger | 29.80 | 1.252 | 1.886 |
| trigger 1 | 0 |  |  |
| trigger 1 | 0 |  |  |
| trigger 2 | 0 |  |  |
| trigger 2 | 0 |  |  |
| trigger 3 | 0 |  |  |
| trigger 3 | 0 |  |  |

**0.020 M IPTG 1 ml**

100 µl 2 M IPTG stock solution

900 µl of MQ H2O

10.08.2022 Team meeting

Present: Evelina, Iida, Jesper, Jonna, Juuli, Kristiina, Malin, Minttu, Nelli

The agenda of this week’s team meeting included discussions about paying the Jamboree team fee, possible sponsors, team photo sessions, and problems in the wet lab.

![A picture of the Zoom window.](https://static.igem.wiki/teams/4207/wiki/aboa-notebook-meeting31.png)

10.08.2022 Trigger plasmids linearization and purification, RNA gel electrophoresis

Present: Evelina, Kristiina, Jesper

**Trigger Plasmid Linearization**

50 µl reactions and 2 for each trigger were made to all triggers, according to our protocol. Incubated for two hours at 37°C.

**Linearized trigger plasmid purification**

Linearised plasmid were purified with NEB Monarch PCR & DNA cleanup kit, according to the kit instructions. The elution was made to 11 µl elution buffer. Concentrations and purity were measured with BioDrop:

| sample | c (ng/µl) | A260/A230 | A260/A280 |
| --- | --- | --- | --- |
| trigger 1 | 49.71 | 1.164 | 1.732 |
| trigger 1 | 44.91 | 1.553 | 1.878 |
| trigger 2 | 39.18 | 1.390 | 1.850 |
| trigger 2 | 38.68 | 1.349 | 1.870 |
| trigger 3 | 37.15 | 1.477 | 1.844 |
| trigger 3 | 45.54 | 1.542 | 1.850 |
| zika trigger | 41.10 | 1.463 | 1.779 |
| zika trigger older | 36.49 | 1.432 | 1.872 |

**RNA gel electrophoresis**

Gel electrophoresis for all the triggers.

Agarose gel was made for the purified Zika V trigger RNA. 50 ml of 1 x TAE buffer was heated with 0.60 g agarose. 10 µl of SYBR safe color was added and mixed. Left to cool.

Samples: 24 µl of glyoxal mix was added to 12 µl of RNA and denatured 50 min in 60℃. 4 µl of loading buffer was added just before loading the gel. The gel was run for 25 min and 15 min after that (Fig. 34). Gel run was successful.

The wells:

| 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 |
| --- | --- | --- | --- | --- | --- | --- | --- |
| ZT 7 | ZT 8 | T1 7 | T1 8 | T2 7 | T2 8 | T3 7 | T2 8 |

![A picture of two agarose gel electrophoresis. On the left hand side, (after 25 min) multiple bands shown in all sample wells except well 2. On the right hand side (after 40 min), the same bands are shown in the sample wells, but a bit fainter, and in some wells, only one band can be seen now.](https://static.igem.wiki/teams/4207/wiki/220810-gel.png)

**Figure 35.**
Agarose gel electrophoresis for ZikaV trigger, triggers 1, 2 and 3 RNA. A. After 25 min of running. B. After 40 min of running.

11.08.2022 Meeting with Patras and TecCEM

11.08.2022 Cell-free reaction with Zika, trigger RNA purification and gel electrophoresis, overnight cultures

Present: Evelina, Jonna, Kristiina, Jesper

**Cell-free reaction**

The following reagents were pipetted to a master mix tube:

| Reagent | Volume (µl) |
| --- | --- |
| AA mix | 13.2 |
| HMP | 1.32 |
| K-G | 1.056 |
| Mg-G | 0.977 |
| Energy mix | 6.6 |
| PEG | 3.3 |
| ONPG | 0.596 |
| Extract | 16.1 |

7.8 μl of the mastermix was transferred to water control and positive control tubes. 4.2 μl of water was added to the water control. 0.521 μl pET15b-LacZ, 0.6 μl 0.02 M IPTG and 3.08 μl of water were added to the positive control tube.

To the mastermix tube, 1.26 µl Zika sensor plasmid was added and 14.4 µl transferred to negative control tube and added 7.6 µl water. To the mastermix tube 6.94 µl water was added and 0.66 µl of trigger RNA.

From water and positive control, 10 μl was loaded into the plate. From both of the sample tubes, 2 x 10 μl reactions were loaded onto the plate as follows:

| 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 | 11 | 12 | 13 | 14 | 15 | 16 | 17 | 18 | 19 | 20 | 21 | 22 | 23 | 24 |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
|  | water | Zika neg | Zika neg |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
|  | pos control | Zika pos | Zika pos |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |

The reactions were incubated at 29°C for 3 hrs, with absorbance measured at 1 min intervals.

![A graph of fluorescent intensity in cell-free reaction measured from different samples presented as a function of time.](https://static.igem.wiki/teams/4207/wiki/220811-cell-free.png)

**Figure 36.**
β-galactosidase activity in cell-free reaction.

**O/N cultures of B69, B46, B39 Toeholds, pEAS002, pET15b-LacZ and ZikaV-Sensor**

5 ml overnight cultures were made with 5 ml LB and the following antibiotics: KAN50 for B39, B46, B69, pEAS002, ZikaV-S and AMP100 for pET15b-LacZ.

**Trigger RNA purification**

Trigger RNA tubes were purified using Monarch total RNA miniprep kit (according to our protocol). Concentrations for tubes (measured with Nanodrop):

| sample | c (ng/µl) | A260/A230 | A260/A280 |
| --- | --- | --- | --- |
| trigger 1 | 296 | 2.444 | 1.972 |
| trigger 1 | 336.2 | 2.523 | 1.941 |
| trigger 2 | 662.3 | 2.535 | 2.005 |
| trigger 2 | 440.6 | 2.629 | 1.962 |
| trigger 3 | 336.5 | 2.640 | 1.997 |
| trigger 3 | 288.2 | 2.591 | 2.026 |
| zika trigger | 412.4 | 2.587 | 1.951 |
| zika trigger | 335.3 | 2.535 | 1.981 |

**RNA gel electrophoresis**

RNAs were denatured for 50 minutes in 60℃ with glyoxal mix according to Juha's protocol.

1,2 % agarose gel was run in 1X TAE buffer for 30 min at 100 V.

![A picture of agarose gel electrophoresis. Faint bands can be seen on all the sample wells, wells 3 and 4 show two bands.](https://static.igem.wiki/teams/4207/wiki/220811-rna-gel.png)

**Figure 37.**
Agarose gel electrophoresis for ZikaV trigger, triggers 1, 2 and 3 RNA. Legend: ZT ZT T1 T1 T2 T2 T3 T3

12.08.2022 Colony PCR for Golden Gate assembled plasmids, plasmid extractions

Present: Jonna, Kristiina, Jesper

**Colony PCR to analyze GG assembled LacZ-toehold plasmids (T7+toehold+LacZ+T7) and MS-toehold A-series plasmids**

2 colonies from each plate were picked for colony PCR (inoculated in 20 µl of MQ-water). Reactions for colony PCR were performed as below (a master mix of primers and Q5 was also made).

Forward primer: toehold linker

Reverse primer: T7 terminator

![A table with reagents on the left side and their concentrations on the right.](https://static.igem.wiki/teams/4207/wiki/220812-pic-1.png)

**Figure 38.**
Concentrations for colony PCR reaction.

PCR reaction:

(40 cycles)

![A table with steps for PCR and temperature and time of each step.](https://static.igem.wiki/teams/4207/wiki/220812-2.png)

**Figure 39.**
PCR cycle for PCR reaction.

Colony PCR was then performed to analyze the results (two colonies from each plasmid).

As can be seen on the gel, none of the LacZ colony PCR reactions show positive results (band size should be 3200 bp), whereas the MS plasmids all have the correct sized band (760 bp).

![A picture of agarose gel electrophoresis. Bands can be seen in sample wells 2, 3, 4, and 5. In well 7 a lot of background is seen. Well 6 and 9 seem empty. Well 11, 12 and 13 show faint bands on the bottom of the gel. Well 14 shows two clear bands and some background. Sample wells 16-21 all show one clear band. Ladders are on wells 1, 8, 15 and 22.](https://static.igem.wiki/teams/4207/wiki/220812-gel.png)

**Figure 40.**
Agarose gel electrophoresis for colony PCR products. Ladder - LacZ\_A70 - LacZ\_A92 - LacZ\_A95 - ladder - LacZ\_B39 - LacZ\_B46 - LacZ\_B69 - ladder - MS\_A70 - MS\_A92 - MS\_A95 - ladder - Neg. control (no DNA.

O/N cultures for MS\_A70 (#1), MS\_A92 (#1) and MS\_A95 (#2) were started.

**Plasmid extractions for B46, B39, B69, pEAS002, pET15b-LacZ, ZikaV-S**

Extraction was made using the NEB Monarch Plasmid Miniprep Kit, according to manufactures’ protocol. First spin was done in 5000 x g 5 min. Samples were eluted in 50 µl of MQ H2O.

Concentrations were as follows:

| Sample | c (ng/µl) | A260/A280 | A260/A230 |
| --- | --- | --- | --- |
| B46 | 176.8 | 1.845 | 1.527 |
| B39 | 161.6 | 1.845 | 1.708 |
| B69 | 133.8 | 1.838 | 1.765 |
| pEAS002 | 108.6 | 1.792 | 0.901 |
| pET15b-LacZ | 214.7 | 1.940 | 1.162 |
| ZikaV-S | 29.97 | 1.877 | 1.111 |

13.08.2022 Wiki writing session

14.08.2022 Media meeting

Present: Anni, Evelina, Jonna, Juuli, Malin, Minttu

We discussed which press and media to contact about our project.

![A picture of the Zoom view.](https://static.igem.wiki/teams/4207/wiki/aboa-notebook-220814media.png)

14.08.2022 Starter culture for ZikaV sensor

Present: Jesper

5 ml of LB was supplemented with 5 µl on kanamycin stock, yielding a final concentration of 50 μg/ml. Single colony of DH5α with Zika-S plasmid was inoculated and incubated overnight at 220 rpm, 37℃.

15.08.2022 Plasmid extractions, PCR reaction for pET15b-LacZ, gel electrophoresis and extraction for PCR products, transformations for three toeholds and pEAS002

Present: Evelina, Anni, Jesper

**Plasmid extraction for ZikaV sensor, MS A70, MS A92, MS A95**

Extraction was made using the NEB Monarch Plasmid Miniprep Kit. First spin was done in 5000 x g 5 min.

Concentrations:

| sample | Concentration (ng/µl) | A260/A230 | A260/A280 |
| --- | --- | --- | --- |
| ZikaV sensor | 268.4 | - | - |
| A70 | 556.4 | 0.867 | 1.890 |
| A9  2 | - | - | - |
| A95 | 294.8 | 1.490 | 2.036 |

**PCR reaction for pet15b-LacZ**

Four reactions and one water control, where DNA was replaced with MQ H2O, was made according to following:

| reagent | volume (µl) |
| --- | --- |
| Master mix | 12.5 |
| forvard primer | 1.25 |
| reverse primer | 1.25 |
| DNA | 1 |
| MQ H2O | 9 |

Following PCR cycle was run:

| 1. | 98 ℃ | 30s |  |
| --- | --- | --- | --- |
| 2. | 98 ℃ | 10s |  |
| 3. | 72 ℃ | 1 min | Highlighted steps were repeated  x 40 |
| 4. | 72 ℃ | 2 min |  |
| 5. | 4 ℃ | ∞ |  |

**Gel electrophoresis for PCR products**

60 ml of 1 x TAE buffer was heated with 0.60 g agarose. 6 µl of SYBR safe color was added and mixed. Left to cool. Poured onto a gel tray.

Samples: 25 µl of each PCR product (4+ neg. control) was mixed with 5 µl of loading Dye and injected to the wells. Also 7 µl of ladder was injected to two wells.

**Gel extraction for PCR products**

Gel extraction was made with the NEB gel extraction kit according to the protocol. The concentrations were measured with Biodrop.

| sample | Concentration (ng/µl) |
| --- | --- |
| 1 | 2.496 |
| 2 | 5.336 |
| 3 | 0.512 |
| 4 | 1.488 |

**Heat-shock transformation for A70+MS, A92+MS, A95+MS and pEAS002**

Two preps of competent DH5a cells were thawed on ice for 15 min. One prep was split in half into two tubes 5 µl of B39\_MS, B46\_MS, and B69\_MS separate tubes. They were incubated on ice for 30 min. Cells were heat-shocked in a 42℃ water bath for 45 s and kept on ice for 7 min. Then, 600 µl of LB was added to each of the tubes and they were placed on 37℃, 200 rpm for 1 hr. From each of the tubes, 100 µl was plated on a LBA + Kan 50 plate. They were incubated at 37℃ overnight.

16.08.2022 Meeting with Aalto-Helsinki + Photography session

16.08.2022 Golden Gate assembly and transformation for three toeholds, colony PCR, trigger plasmid extractions, ZikaV trigger plasmid linearization, purification and RNA synthesis

Present: Evelina, Jonna, Anni, Kristiina, Kevät, Jesper

**Golden gate assembly for T7 + mScarlet-I**

3 reactions were made, one for each toehold. All the concentrations were as before (18.7.), but the concentration of the reporter PCR product is 20.6 ng/µl. The components for each of the reactions are:

| pODD1 (µl) | T7 promoter | Toehold | LacZ | Terminator | Buffer | Enzyme | H2O |
| --- | --- | --- | --- | --- | --- | --- | --- |
| 1 | 0.31 | A92 | 0.29 | 3.6 | 0.32 | 2 | 1 | 11.48 |
| 1 | 0.31 | A95 | 0.24 | 3.6 | 0.32 | 2 | 1 | 11.53 |
| 1 | 0.31 | B39 | 0.30 | 3.6 | 0.32 | 2 | 1 | 11.47 |
|  |  |  |  |  |  |  |  |  |

Golden gate reaction protocol (according to instructions on NEB site): (1 min 37℃, 1 min 16℃) X 30, 5 min 60℃, keep on 4℃. Stored at -20℃.

**Heat-shock transformation for A92, B39 and A95**

Three preps of competent DH5a cells were thawed on ice for 15 min. 7 µl of A92, B39 and A95 were added into separate tubes. They were incubated on ice for 30 min. Cells were heat-shocked in a 42℃ water bath for 45 s and kept on ice for 7 min. Then, 700 µl of LB was added to each of the tubes and they were placed on 37℃, 200 rpm for 1 hr. From each of the tubes, 100 µl and 300 µl was plated on a LBA + Kan 50 plate. They were incubated at 37℃ overnight.

**Colony PCR for MS\_B-toeholds**

| Reaction components | 175 µl |
| --- | --- |
| Q5 High Fidelity 2x MasterMix | 87.5 µl |
| 10 µM F Toehold Linker | 8.75 µl |
| 10 µM R T7 Terminator | 8.75 µl |
| MQ Water | 70 µl |

7 x MasterMix was done according to the table above. Distributed into seven PCR tubes, 25 µl to each. Two colonies were selected from each plate (2 x B69, 2 x B46, 2 x B39). A streak of each plate was inoculated into the reaction mixtures. Run according to the table below:

| 1. Initidial Denaturation | 98℃ 30 s |
| --- | --- |
| 2. Cycle 40 X | a) 98℃ 10 s |
|  | b) 72℃ 30 s |
|  | c) 72℃ 30 s |
| 3. Final Extension | 72℃ 2 min |
| 4. Hold | Infinity |

1 % agarose gel was run at 100 V for 35 minutes.

![A picture of agarose gel electrophoresis. One clear band can be seen in sample wells 2-7 below 1 kb. Sample wells 10 and 11 both show one faint band.](https://static.igem.wiki/teams/4207/wiki/220816-gel.png)

**Figure 41.**
Agarose gel electrophoresis for colony PCR products. Wells: 1 kb DNA Ladder, B39 1, B39 2, B46 1, B46 2, B69 1, B69 2, Negative Control, 1 kb DNA Ladder, Linearised Zika V trigger, Linearised Zika V trigger.

**Plasmid extraction for ZikaV trigger, trigger 1, trigger 2, trigger 3, pET15b-LacZ**

Extraction was made using NEB Monarch Plasmid Miniprep Kit. First spin was done in 3000 x g 5 min.

Concentrations (measured with BioDrop):

| sample | c (ng/µl) | A260/A230 | A260/280 |
| --- | --- | --- | --- |
| trigger 1 | 56.48 | 0.598 | 1.687 |
| trigger 2 | 247.8 | 2.069 | 1.852 |
| trigger 3 | 13.47 | 1.080 | 1.804 |
| zika trigger | 114.7 | 1.623 | 1.989 |
| pET15b-LacZ | 114.7 | 1.623 | 1.989 |

**Zika trigger Plasmid Linearization**

2 50 µl reactions were made, reagents: 1 µl AvrII, 5 µl rCutSmart, 35.28 µl MQ H2O, 8.72 µl DNA. Incubated for two hours at 37°C.

**Linearized trigger plasmid purification**

Linearized plasmid were purified with NEB Monarch PCR & DNA cleanup kit, according to the kit instructions. The elution was made to 15 µl elution buffer. Concentrations and purity were measured with BioDrop:

| sample | c (ng/µl) | A260/A230 | A260/280 |
| --- | --- | --- | --- |
| 1 | 15.54 | 0.689 | 1.474 |
| 2 | 14.43 | 1.00 | 1.712 |

**Zika V trigger RNA synthesis**

3 RNA synthesis reactions were made. The reagents were pipetted in the tubes in following order: 1.5 µl of RNAP reaction buffer; 1.5 µl of each of the NTPs; 8 µl of linearised DNA; 1.5 µl of T7 RNAP. (17 µl total) The reactions were incubated at 37℃ overnight.

17.08.2022 Team meeting

Present: Evelina, Iida, Jesper, Jonna, Juuli, Kristiina, Minttu, Nelli, Sofia

We updated each other about all the things that happened during the week as well as discussed wiki text responsibilities. We also took a look at the judging forms from the Nordic iGEM Conference and tried to learn things we could improve in our upcoming presentations!

![A picture of the Zoom window.](https://static.igem.wiki/teams/4207/wiki/aboa-notebook-meeting32.png)

17.08.2022 Cell-free reaction with Zika, Golden Gate reactions and transformations, ZikaV trigger plasmid linearization, purification and RNA synthesis

Present: Evelina, Jesper

**Cell-free reaction**

The following reagents were pipetted to a master mix tube:

| Reagent | Volume (µl) |
| --- | --- |
| AA mix | 13.2 |
| HMP | 1.32 |
| K-G | 1.056 |
| Mg-G | 0.977 |
| Energy mix | 6.6 |
| PEG | 3.3 |
| ONPG | 0.596 |
| Extract | 16.1 |

7.8 μl of the mastermix was transferred to water contol and positive control tubes. 4.2 μl of water was added to the water control. 0.521 μl pET15b-LacZ, 0.6 μl 0.02 M IPTG and 3.08 μl of water were added to the positive control tube.

To the mastermix tube, 1.26 µl Zika sensor plasmid was added and 14.4 µl transferred to negative control tube and added 7.6 µl water. To the mastermix tube 6.94 µl water was added and 0.66 µl of trigger RNA.

From water and positive control, 10 μl was loaded into the plate. From both of the sample tubes, 2 x 10 μl reactions were loaded onto the plate as follows:

| 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 | 11 | 12 | 13 | 14 | 15 | 16 | 17 | 18 | 19 | 20 | 21 | 22 | 23 | 24 |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
|  | water | Zika neg | Zika pos |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
|  | pos control | Zika neg | Zika pos |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |

The reactions were incubated at 29°C for 3 hrs, with absorbance measured at 1 min intervals.

![A graph of absorbance measured in cell-free reaction as a function of time with different samples.](https://static.igem.wiki/teams/4207/wiki/220817-cell-free.png)

**Figure 42.**
β-galactosidase activity in cell-free reaction.

Here, even our positive control did not produce anything, we think this is because of the amino acid mix.

**Golden gate reactions**

6 reactions were made, one for each toehold. All the concentrations were as before (18.7.), but the concentration of the reporter PCR product is 20.6 ng/µl. The components for each of the reactions are:

| pODD1 (µl) | T7 promoter | Toehold | LacZ | Terminator | Buffer | T4 | BsaI-HF v2 | H2O |
| --- | --- | --- | --- | --- | --- | --- | --- | --- |
| 1 | 0.31 | B69 | 0.27 | 8.2 | 0.32 | 2 | 0.25 | 0.75 | 6.9 |
| 1 | 0.31 | B46 | 0.31 | 8.2 | 0.32 | 2 | 0.25 | 0.75 | 6.86 |
| 1 | 0.31 | A70 | 0.29 | 8.2 | 0.32 | 2 | 0.25 | 0.75 | 6.88 |
| 38.49 | 12.83 |  |  |  |  |  |  |  |  |

Golden gate reaction protocol (according to instructions on NEB site): (1 min 37℃, 1 min 16℃) X 30, 5 min 60℃, keep on 4℃. Stored at -20℃.

**Heat-shock transformation for golden gate assembled plasmids**

Two preps of competent DH5a cells were thawed on ice for 15 min. Approximately 33.3 µl from both tubes was transferred to a new one. 5 µl DNA was added and incubated on ice for 30 min. Cells were heat-shocked in a 42℃ water bath for 45 s and kept on ice for 5 min. Then, 500 µl of LB was added to each of the tubes and they were placed on 37℃, 200 rpm for 1 hr. From each of the tubes, 100 µl was plated on an LBA + Kan 50 plate. They were incubated at 37℃ overnight.

**Zika trigger Plasmid Linearization**

One 50 µl reaction was made, reagents: 1 µl AvrII, 5 µl rCutSmart, 35.28 µl MQ H2O, 8.72 µl DNA. Incubated for one hour at 37°C.

**Linearised trigger plasmid purification**

Linearised plasmid was purified with NEB Monarch PCR & DNA cleanup kit, according to the kit instructions. The elution was made to 12 µl elution buffer. Concentration and purity was measured with BioDrop: c=20.08 ng/µl, A260/A230=0.953; A260/A280=1.426.

**Zika V trigger RNA synthesis**

The reagents were pipetted in the tubes in following order: 1.5 µl of RNAP reaction buffer; 1.5 µl of each of the NTPs; 11 µl of linearised DNA; 1.5 µl of T7 RNAP. (20 µl total) The reactions were incubated at 37℃ overnight.

18.08.2022 Human Practices meeting + Meeting with Tirupati + Meeting with Patras and TecCEM

Present: Anni, Jesper, Kevät, Kristiina, Malin, Nelli

The main discussion themes included educational material, interviews, and the upcoming Study in Turku fair.

![A picture of the Zoom view.](https://static.igem.wiki/teams/4207/wiki/aboa-notebook-220818hp.png)

18.08.2022 Cell-free reaction with Zika, colony PCR, trigger plasmid linearization and purifications, ssDNA trigger resuspensions

Present: Evelina, Kristiina, Jesper

**Cell-free reaction**

The reaction protocol from yesterday was repeated, but new AA mix was used.

The samples were added to the plate in the following order:

| 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 | 11 | 12 | 13 | 14 | 15 | 16 | 17 | 18 | 19 | 20 | 21 | 22 | 23 | 24 |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
|  |  |  |  | Water | Neg. sample | Pos. sample |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
|  |  |  |  | Pos. control + Neg. sample |  | Pos. sample |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |

The other neg. sample was added to the positive control well by accident.

![A graph of absorbance measured in cell-free reaction as a function of time with different samples.](https://static.igem.wiki/teams/4207/wiki/220818-cell-free.png)

**Figure 43.**
β-galactosidase activity in cell-free reaction.

**ColonyPCR for Golden Gate assembled toehold plasmids from 17th and 16th of August**

| Reaction components | Volume |  | PCR program | Temperature | Time |
| --- | --- | --- | --- | --- | --- |
| Reaction volume | 350 µl |  | Initial denaturation | 98℃ | 30 s |
| Q5 High Fidelity 2x MasterMix | 175 µl |  | Cycle 40X | a) 98℃ | 10 s |
| 10 µM F Toehold Linker | 17.5 µl |  |  | b) 72℃ | 1 min |
| 10 µM R T7 Terminator | 17.5 µl |  | Final extension | 72℃ | 2 min |
| MQ Water | 140 µl |  | Hold | 4℃ | ∞ |

14X MasteMix was made according to the table above. It was then divided into 13 PCR tubes, 25 µl each. Each tube was inoculated with a colony from a plate: 2 tubes for each plate (B39, B46, B69, A70, A92, A95), except for the water control. The PCR reaction was run according to the table above.

**Gel electrophoresis for colonyPCR products**

25 µl samples were made adding 5 µl Purple Loading Dye (NEB) to the PCR products.

After 35 min 100V. Upper row: 2: Ladder 3: A70 I 4: A70 II 5: A92 I 6: A92 II 7: A95 I 8: A95 II 9: H2O. Lower row: 2: Ladder 3:B39 I 4: B39 II 5: B46 I 6: B46 II 7: B69 I 8: B69 II

After 50 min 100V. Upper row: 2: Ladder 3: A70 I 4: A70 II 5: A92 I 6: A92 II 7: A95 I 8: A95 II 9: H2O.

After 50 min 100 V. Lower row: 2: Ladder 3:B39 I 4: B39 II 5: B46 I 6: B46 II 7: B69 I 8: B69 II

![A picture of 4 agarose gel electrophoresis. In figure A, Clear bands can be seen on sample wells 5, 7 and 8 (different sizes). In wells 2-4 and 6 a lot of background can be seen, with a smudgy band on the bottom of the gel. In figure B same bands as in figure A can be seen. In figure C, A lot of background can be seen in all the sample wells, with some smudgy bands on the bottom of the gel in most of the wells. In figure D, one band can be seen on well 2, otherwise very faint background of nothing on the wells.](https://static.igem.wiki/teams/4207/wiki/220818-gel.png)

**Figure 44.**
Agarose gel electrophoresis for colony PCR products. A. Upper row of the gel after 35 min running. B. Lower row of the gel after 35 min running. C. Upper row of the gel after 50 min running. D. Lower row of the gel after 50 min running.

There is a potential contamination. A92 II, A95 II and B39 I might be alright.

**Trigger Plasmid Linearization**

4 50 µl reaction was made, reagents: 1 µl AvrII(for Zika trigger)/Pst1(for others), 5 µl rCutSmart, 1 µg DNA (ZikaV-T: 8.75 µl; T1: 17.71 µl; T2: 4.04 µl; T3: 44 µl), MQ H20 to 50 µl (ZikaV-T: 35.28 µl; T1: 26.29 µl; T2: 39,.96 µl; T3: 0 µl). Incubated for one hour at 37°C and 20 min at 80℃.

**Linearised trigger plasmid purification**

Linearised plasmid was purified with NEB Monarch PCR & DNA cleanup kit, according to the kit instructions. The elution was made to 12 µl elution buffer. Concentration and purity was measured with BioDrop:

| Sample | c (ng/µl) | A260/A230 | A260/A280 |
| --- | --- | --- | --- |
| ZikaV-T | 15.95 | 1.335 | 1.782 |
| T1 | 18.40 | 0.948 | 1.769 |
| T2 | 61.85 | 1.634 | 1.827 |
| T3 | 38.77 | 1.348 | 1.781 |

**Trigger ssDNA resuspension**

100 µM resuspensions were made of each trigger ssDNA (Trigger B46; Trigger A70,B69; Trigger A92,A95) as follows:

| Trigger | n (nmol) | V(H20) µl |
| --- | --- | --- |
| B46 | 25.4 | 254 |
| A70, B69 | 19.7 | 197 |
| A92, A95 | 19.9 | 199 |

After this 200 µl 10 µM dilutions of each of the ssDNAs were made by adding 180 µl MQ H20 to 20 µl ssDNA. Both solutions were stored in -20℃.

19.08.2022 Meeting with Aalto-Helsinki

19.08.2022 Cell-free reaction with ssDNA triggers, protein concentration measurements for purified mScarlet-I and β-galactosidase

Present: Evelina, Kristiina, Jesper

**Cell-free reaction**

The following reagents were pipetted to a master mix tube:

| Reagent | Volume (µl) |
| --- | --- |
| AA mix | 22.4 |
| HMP | 2.24 |
| K-G | 1.792 |
| Mg-G | 1.658 |
| Energy mix | 11.2 |
| PEG | 5.6 |
| Extract | 27.32 |

7.8 μl of the master mix was transferred to water control and positive control tubes. 4.2 μl of water was added to the water control. 1.636 μl pEAs002; 0.6 μl 0.02 M IPTG and 1.964 μl of water were added to the positive control tube.

28.3 µl was transferred from the mastermix tube to another tube. 1.726 µl A92 sensor was added and 15.013 µl transferred to negative control tube and added 6.987 µl H2O. To the another tube, 5.887 µl water and 1,1 µl ssDNA trigger 1.

To the mastermix tube, 1.17 μl A70 sensor plasmid was added and 14.735 μl transferred to negative control tube and added 7.265 μl water. To the mastermix tube 6.165 μl water was added and 1.1 μl of ssDNA trigger 2.

From water and positive control, 10 μl was loaded into the plate. From all of the sample tubes, 2 x 10 μl reactions were loaded onto the plate as follows:

| 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 | 11 | 12 | 13 | 14 | 15 | 16 | 17 | 18 | 19 | 20 | 21 | 22 | 23 | 24 |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
|  | water | A70 neg | A70 pos | A92 neg | A92 pos |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
|  | pos control | A70 neg | A70 pos | A92 neg | A92 pos |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |

The reactions were incubated at 29°C for 3 hrs, with fluorescence measured at 1 min intervals.

![A graph of fluorescent intensity in cell-free reaction measured from different samples presented as a function of time.](https://static.igem.wiki/teams/4207/wiki/220819-cell-free.png)

**Figure 45.**
mScarlet-I produced in cell-free reaction.

**Protein concentration measurements for purified mScarlet-I and β-galactosidase**

1 ml samples of BSA with concentrations between 0 and 10 μg/ml were prepared. 1/300 dilution of mScarlet-I and 1/30 dilution of β-galactosidase were prepared to 1 ml volume. 250 µl of Bradford dye working solution was added to each sample and they were incubated for 5 min. Each sample's absorbance was measured at 595 nm and a standard curve was

20.08.2022 Wiki writing session + Wellbeing day

22.08.2022 Cell-free reaction with ssDNA triggers, overnight cultures for some of the toeholds

Present: Evelina, Kristiina

**Cell-free reaction**

The following reagents were pipetted to a master mix tube:

| Reagent | Volume (µl) |
| --- | --- |
| AA mix | 13.6 |
| HMP | 1.36 |
| K-G | 1.09 |
| Mg-G | 1.00 |
| Energy mix | 6.8 |
| PEG | 3.4 |
| ONPG | 0.61 |
| Extract | 16.58 |

7.8 μl of the master mix was transferred to water control and positive control tubes. 4.2 μl of water was added to the water control. 0.521 μl pET15b-LacZ, 0.6 μl 0.02 M IPTG and 3.08 μl of water were added to the positive control tube.

To the mastermix tube, 1.31 μl Zika sensor plasmid was added and 15.1 μl transferred to negative control tube and added 6.9 μl water. To the mastermix tube 5.9 μl water was added and 1.1 μl of ssDNA trigger.

From water and positive control, 10 μl was loaded into the plate. From both of the sample tubes, 2 x 10 μl reactions were loaded onto the plate as follows:

| 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 | 11 | 12 | 13 | 14 | 15 | 16 | 17 | 18 | 19 | 20 | 21 | 22 | 23 | 24 |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
|  |  |  |  |  |  |  | water | Zika neg | Zika pos |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
|  |  |  |  |  |  |  | pos control | Zika neg | Zika pos |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |

The reactions were incubated at 29°C for 3 hrs, with absorbance (420 nm) measured at 1 min intervals.

![A graph of absorbance measured in cell-free reaction as a function of time with different samples.](https://static.igem.wiki/teams/4207/wiki/220822-cell-free.png)

**Figure 46.**
β-galactosidase activity in cell-free reaction.

**O/N cultures of A95-LacZ, A92-LacZ, B39-LacZ and B39-mS, B46-mS, B69-mS**

5 ml LB KAN50 overnight cultures of A95-LacZ, A92-LacZ, B39-LacZ and B39-mS, B46-mS, B69-mS were made.

23.08.2022 Meeting with Tirupati

23.08.2022 Cell-free reaction with Zika, plasmid extractions

Present: Evelina, Jonna, Kristiina

**Cell-free reaction with Zika V**

The following reagents were pipetted to a master mix tube:

| Reagent | Volume (µl) |
| --- | --- |
| AA mix | 25.8 |
| HMP | 2.58 |
| K-G | 2.064 |
| Mg-G | 1.909 |
| Energy mix | 12.9 |
| PEG | 6.45 |
| ONPG | 1.157 |
| Extract | 31.463 |

7.8 µl was transferred to water control and 4.2 µl water added. 11.7 µl was transferred to pos control tube, 0.782 µl pET15b-LacZ; 0.9 µl 0.02 M IPTG and 4.62 µl water added.

21.6 µl was transferred to other tubes (6 and 9). To the mastermix tube 2.943 µl Zika Sensor was added, then divided into three tubes. One was added 2.412 µl water and 0.675 µl trigger RNA, on 2.479 µl water and 0.338 µl trigger RNA and last 2.816 µl water.

To the tube 6 4.414 µl Zika Sensor was added, then divided into three tubes. One was added 1.651 µl water and 0.675 µl trigger RNA, on 1.988 µl water and 0.338 µl trigger RNA and last 2.326 µl water.

To the tube 9 5.885 µl Zika Sensor was added, then divided into three tubes. One was added 1.179 µl water and 0.675 µl trigger RNA, on 1.498 µl water and 0.338 µl trigger RNA and last 1.836 µl water.

From pos control tube one 5 µl and one 10 µl reaction was loaded into plate and from all others 10 µl was loaded into plate as follows:

| 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 | 11 | 12 | 13 | 14 | 15 | 16 | 17 | 18 | 19 | 20 | 21 | 22 | 23 | 24 |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
|  |  |  |  |  |  |  |  |  |  | water | 6 nM + 500 nM trigger | 9 nM + 500 nM trigger | 12 nM + 500 nM trigger |  |  |  |  |  |  |  |  |  |  |
|  |  |  |  |  |  |  |  |  |  | pos control 5 µl | 6 nM + 1000 nM trigger | 9 nM + 1000 nM trigger | 12 nM + 1000 nM trigger |  |  |  |  |  |  |  |  |  |  |
|  |  |  |  |  |  |  |  |  |  | pos control 10 µl | 6 nM neg | 9 nM neg | 12 nM neg |  |  |  |  |  |  |  |  |  |  |
|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |

The reactions were incubated at 29°C for 3 hrs, with absorbance (420 nm) measured at 1 min intervals.

![A graph of absorbance measured in cell-free reaction as a function of time with different samples.](https://static.igem.wiki/teams/4207/wiki/220823-cell-free.png)

**Figure 47.**
β-galactosidase activity in cell-free reaction.

**Plasmid extraction for the minipreps A95-LacZ, A92-LacZ, B39-LacZ and B39-mS, B46-mS, B69-mS**

Plasmid extraction with Thermo Scientific GeneJET plasmid miniprep was made for A95-LacZ, A92-LacZ, B39-LacZ and B39-mS, B46-mS, B69-mS.

24.08.2022 Team meeting + Meeting with Aalto-Helsinki

Present: Evelina, Jonna, Juuli, Kevät, Kristiina, Malin, Minttu, Nelli

We started our crowdfunding campaign and wrote texts for it. We also discussed the upcoming fair in which we were going to be exhibitors, and shared some wiki text responsibilities.

![A picture of the Zoom window.](https://static.igem.wiki/teams/4207/wiki/aboa-notebook-meeting33.png)

24.08.2022 Cell-free reaction with Zika

Present: Evelina, Kristiina

**Cell-free reaction sonication ZikaV toeh 2 nM, 6 nM + RNA 1500 nM + vortex test**

The following reagents were pipetted to a master mix tube 4:

| Reagent | Volume (µl) |
| --- | --- |
| AA mix | 24.8 |
| HMP | 2.48 |
| K-G | 1.984 |
| Mg-G | 1.835 |
| Energy mix | 12.4 |
| PEG | 6.2 |
| ONPG | 1.112 |
| Extract | 30.244 |

7.8 µl of the master mix was transferred to the water control tube (1) and 4.2 µl of water was added. 15.6 µl of the master mix was transferred to the positive control tube (2), 1.042 µl pET15b-LacZ; 1.2 µl 0.02 M IPTG and 6,16 µl water were added. 12 µl was transferred to tube 3 and vortexed for 30 s.

28.8275 µl of the master mix was transferred to tube 8, and 3.924 µl of ZikaV-Sensor plasmid was added there. 16.3758 µl of this was transferred to tube 10 and 5.624 µl of water was added there. 11 µl of this was transferred into a new tube (11) and vortexed for 30 s. Into tube 8 was added 2.026 µl of ZikaV-Trigger RNA and 3.598 µl of water. 11 µl of this was transferred to tube 9 and vortexed for 30 s.

Into tube 4 was added 1.308 µl of ZikaV-Sensor plasmid and 15.068 µl was transferred to tube 6. Into tube 6 was added 6.932 µl of water and 11 µl of this was vortexed for 30 s (tube 7). After this into tube 4 was added 2.026 µl ZikaV-Trigger RNA and 4.906 µl of water. 11 µl of this was transferred into tube 5 and vortexed for 30 s.

From every tube, one 10 µl sample was loaded to a clear 384 well plate in the following order:

| Well | Sample | Tube no. |
| --- | --- | --- |
| E15 | Water control | 1 |
| E16 | Positive control, no vortex | 2 |
| E17 | Positive control, with vortex | 3 |
| F15 | 2 nM ZikaV-Sensor, RNA | 4 |
| F16 | 2 nM ZikaV-Sensor, RNA, with vortex | 5 |
| F17 | 6 nM ZikaV-Sensor, RNA | 8 |
| F18 | 6 nM ZikaV-Sensor, RNA, with vortex | 9 |
| G15 | 2 nM ZikaV-Sensor | 6 |
| G16 | 2 nM ZikaV-Sensor, with vortex | 7 |
| G17 | 6 nM ZikaV-Sensor | 10 |
| G18 | 6 nM ZikaV-Sensor, with vortex | 11 |

The reactions were incubated at 29°C for 3 hrs, with absorbance (420 nm) being measured at 1 min intervals.

![A graph of absorbance measured in cell-free reaction as a function of time with different samples.](https://static.igem.wiki/teams/4207/wiki/220824-cell-free.png)

**Figure 48.**
β-galactosidase activity in cell-free reaction.

25.08.2022 Cell-free reactions with A70 and A92 mScarlet-I toeholds and ZikaV

Present: Evelina, Kristiina

**Cell free with mScarlet 3h**

The following reagents were pipetted to a master mix tube 5:

| Reagent | Volume (µl) |
| --- | --- |
| AA mix | 31.2 |
| HMP | 3.12 |
| K-G | 2.496 |
| Mg-G | 2.3088 |
| Energy mix | 15.6 |
| PEG | 7.8 |
| Extract | 38.05 |

7.8 µl of the master mix was transferred to the water control tube 1 and 4.2 µl of water was added. 7.8 µl of the master mix was transferred to positive control tube 2, 1.636 µl pEAS002; 0.6 µl 0.02 M IPTG and 1.964 µl water were added.

21.24 µl of the master mix was transferred to tube 12, and 3.349 µl of A92 toehold plasmid and 1.65 µl IPTG were added there. 8.747 µl of this was transferred to tube 13 and 1.152 µl of water and 1.1 µl trigger 1 ssDNA added there. 8.747 µl from tube 12 was transferred to tube 14 and 2.252 µl water was added. 2.252 µl of trigger 1 RNA was added to tube 12.

21.24 µl of the master mix was transferred to tube 9, and 1.675 µl of A92 toehold plasmid and 1.65 µl IPTG were added there. 8.19 µl of this was transferred to tube 10 and 1.71 µl of water and 1.1 µl trigger 1 ssDNA added there. 8.19 µl from tube 11 was transferred to tube 14 and 2.81 µl water was added. 2.81 µl of trigger 1 RNA was added to tube 12.

21.24 µl of the master mix was transferred to tube 6, and 2.272 µl of A70 toehold plasmid and 1.65 µl IPTG were added there. 8.389 µl of this was transferred to tube 7 and 1.511 µl of water and 1.1 µl trigger 1 ssDNA added there. 8.389 µl from tube 8 was transferred to tube 14 and 2.611 µl water was added. 1.602 µl of trigger 2 RNA and 1.009 µl of water were added to tube 6.

To tube 5 1.136 µl of A70 toehold plasmid and 1.65 µl IPTG were added. 8.00 µl of this was transferred to tube 4 and 1.9 µl of water and 1.1 µl trigger 1 ssDNA added there. 8.0 µl from tube 5 was transferred to tube 3 and 3.00 µl water was added. 1.602 µl of trigger 2 RNA and 1.398 µl of water were added to tube 5.

From every tube, one 10 µl sample was loaded to a black 384 well plate in the following order:

| Well | Sample | Tube no. |
| --- | --- | --- |
| B7 | Water control | 1 |
| B8 | Positive control pEAS002 | 2 |
| B9 | 10 nM Toehold A70 | 3 |
| B10 | 10 nM Toehold A70, 1500 nM Trigger2 ssDNA | 4 |
| B11 | 10 nM Toehold A70, 1500 nM Trigger2 RNA | 5 |
| C7 | 20 nM Toehold A70, 1500 nM Trigger2 RNA | 6 |
| C8 | 20 nM Toehold A70, 1500 nM Trigger2 ssDNA | 7 |
| C9 | 20 nM Toehold A70 | 8 |
| C10 | 10 nM Toehold A92, 1540 nM Trigger1 RNA | 9 |
| C11 | 10 nM Toehold A92, 1540 nM Trigger1 ssDNA | 10 |
| C12 | 10 nM Toehold A92 | 11 |
| D7 | 20 nM Toehold A92, 1230 nM Trigger1 RNA | 12 |
| D8 | 20 nM Toehold A92, 1230 nM Trigger1 ssDNA | 13 |
| D9 | 20 nM Toehold A92 | 14 |

The reactions were incubated at 29°C for 3 hrs, with fluorescence at 569/593 nm being measured at 1 min intervals. Gmin value was changed manually to 110.

![A graph of fluorescence intensity measured in cell-free reaction as a function of time with different samples.](https://static.igem.wiki/teams/4207/wiki/220825-cell-free-ms.png)

**Figure 49.**
mScarlet-I produced in cell-free reaction.

**Cell free with Zika O/N 12h**

The following reagents were pipetted to a master mix tube 4:

| Reagent | Volume (µl) |
| --- | --- |
| AA mix | 17.2 |
| HMP | 1.72 |
| K-G | 1.376 |
| Mg-G | 1.273 |
| Energy mix | 8.6 |
| PEG | 4.3 |
| ONPG | 0.771 |
| Extract | 20.98 |

3.9 µl of the master mix was transferred to the water control tube 1 and 2.1 µl of water was added. 7.8 µl of the master mix was transferred to positive control tube 2, 0.521 µl pET15b-LacZ; 0.6 µl 0.02 M IPTG and 3.08 µl water were added.

22.26 µl of the master mix was transferred to tube 7, and 3.032 µl of ZikaV Sensor plasmid and 1.7 µl IPTG were added there. 8.99 µl of this was transferred to tube 5 and 0.99 µl of water and 1.013 µl ZikaV trigger RNA added there. 8.99 µl from tube 7 was transferred to tube 6 and 0.653 µl water and 1.35 µl ZikaV Trigger RNA added. 2.00 µl of water was added to tube 7.

3.032 µl of ZikaV Sensor plasmid was added to tube 4. 8.43 µl of this was transferred to tube 8 and 1.56 µl of water and 1.013 µl ZikaV trigger RNA added there. 8.43 µl from tube 4 was transferred to tube 3 and 1.22 µl water and 1.35 µl ZikaV Trigger RNA added. 2.57 µl of water was added to tube 4.

From every tube, one 10 µl sample was loaded to a black 384 well plate in the following order:

| Well | Sample | Tube no. |
| --- | --- | --- |
| E19 | Water control | 1 |
| E20 | Positive control pET15b-LacZ | 2 |
| F19 | 6 nM ZikaV Sensor, 1500 nM ZikaV Trigger | 8 |
| F20 | 6 nM ZikaV Sensor, 2000 nM ZikaV Trigger | 3 |
| F21 | 6 nM ZikaV Sensor | 4 |
| G19 | 6 nM ZikaV Sensor, 1500 nM ZikaV Trigger, IPTG | 5 |
| G20 | 6 nM ZikaV Sensor, 2000 nM ZikaV Trigger, IPTG | 6 |
| G21 | 6 nM ZikaV Sensor, IPTG | 7 |

The reactions were incubated at 29°C for 3 hrs, with absorbance (420 nm) being measured at 1 min intervals.

![A graph of absorbance measured in cell-free reaction as a function of time with different samples.](https://static.igem.wiki/teams/4207/wiki/220825-cell-free-lacz.png)

**Figure 50.**
β-galactosidase activity in cell-free reaction.

27.08.2022 Wiki writing session

29.08.2022 Wet Lab meeting

Present: Kevät, Jonna, Evelina, Anni, Jesper, Sofia

The meeting consisted of troubleshooting and discussions about resources that are still left to use.

![A picture of the Zoom view.](https://static.igem.wiki/teams/4207/wiki/aboa-notebook-220829wetlab.png)

30.08.2022 Study in Turku fair

![People organizing a workshop.](https://static.igem.wiki/teams/4207/wiki/aboa-notebook-studyinturku.jpg)

31.08.2022 Team meeting

Present: Jonna, Juuli, Kevät, Minttu, Nelli

We agreed on a new time for our weekly meetings as well as discussed our collaborations and partnerships. Wiki texts responsibilities were also negotiated and the importance of schedules and internal deadlines was emphasized.

![A picture of the Zoom window.](https://static.igem.wiki/teams/4207/wiki/aboa-notebook-meeting34.png)

SEP

01.09.2022 Sponsor meeting + Meeting with Patras and TecCEM

01.09.2022 Cell-free reaction with RNA synthesis components, ZikaV trigger linearisation, purification and gel electrophoresis

Present: Jonna, Anni, Jesper

**Cell-free reaction**

The amounts of reagents were calculated using the master mix calculator, with 80 μl as the final volume. The following master mix was created to tube 4:

| Reagent | Volume(μl) |
| --- | --- |
| AA mix | 16 |
| HMP | 1.6 |
| K-G | 1.28 |
| Mg-G | 1.184 |
| Energy mix | 8 |
| PEG | 4 |
| ONPG | 0.722 |
| Extract | 19.5 |

7.8 μl of this master mix was transferred to tube 1 (water control) and tube 2 (positive control). 4.2 μl of water was added to tube 1, while 1.1 μl pET15b-LacZ, 0.6 μl 0.02 M IPTG and 2.5 μl of water was added to tube 2.

22.25 μl of the master mix was transferred to tube 5 and it was supplemented with 1.7 μl of T7 RNAP and 1.5 μl of each of the NTP solutions.

1.96 μl and 3 μl of Zika-S plasmid was added to tubes 4 and 5 respectively.

7.2 μl of the mix from tube 4 and 3.8 μl of water was added to tube 3 (negative sample). 1.1 μl of purified RNA trigger and 2.7 μl of water was added to tube 4 (positive sample + RNA)

9.7 μl of the mix from tube 5 and 1.3 μl of water was added to tube 6 (negative sample + RNA synthesis components). 2.8 μl of the linearised DNA was added to tube 5 (positive sample + RNA synthesis).

The samples were loaded onto a clear-welled 384-well plate in the following order:

| 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 | 11 | 12 | 13 | 14 | 15 | 16 | 17 | 18 | 19 | 20 | 21 | 22 | 23 | 24 |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
|  | Water control | Neg. sample | pos. sample + RNA | pos. sample + RNA synthesis |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
|  | pos. control | neg. sample + synthesis components |  | pos. sample + RNA synthesis |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |

The reactions were incubated at 37°C for 6 hrs, with absorbance at 420 nm measured at 1 min intervals.

![A graph of absorbance measured in cell-free reaction as a function of time with different samples.](https://static.igem.wiki/teams/4207/wiki/220901-cell-free.png)

**Figure 51.**
β-galactosidase activity in cell-free reaction. The measurement settings were set up inaccurately, so only the bottom three wells were measured.

Here, we can clearly see that the positive sample produced β-galactosidase and it produced more than the negative sample. After this, we continued the measurement for another 6 hrs, measuring all the sample wells.

![A graph of absorbance measured in cell-free reaction as a function of time with different samples.](https://static.igem.wiki/teams/4207/wiki/220901-cell-free-2.png)

**Figure 52.**
β-galactosidase activity in cell-free reaction.

Here, the RNA-synthesis sample as well as the negative sample with synthesis components continue to produce

**Zika-T Linearisation**

8.7 µl of Zika-T plasmid, 5 µl of rCutSmart buffer, 1 µl of AvrII enzyme and 35.3 µl of water were added to a tube. The digestion reaction was incubated for 1 hr. 7.5 µl of this reaction mixture was mixed with 1.5 µl of 6x purple loading dye and gel electrophoresis was performed at 100 V for 45 min.

![A picture of agarose gel electrophoresis. A bright band can be seen in the sample well.](https://static.igem.wiki/teams/4207/wiki/220901-gel.png)

**Figure 53.**
Agarose gel electrophoresis for linearised ZikaV trigger plasmid.

**Product purification**

The rest of the product was purified using the Monarch PCR & DNA cleanup kit, with the final suspension done with 6 µl.

02.09.2022 Cell-free reaction with A70 mScarlet-I

Present: Jesper

**Cell-free reaction**

The amounts of reagents were calculated using the master mix calculator, with 112 μl as the final volume. The following master mix was created to tube 4:

| Reagent | Volume(μl) |
| --- | --- |
| AA mix | 16 |
| HMP | 1.6 |
| K-G | 1.28 |
| Mg-G | 1.184 |
| Energy mix | 8 |
| PEG | 4 |
| Extract | 19.5 |

7.8 μl of the mastermix was transferred to tubes 1 and 2. 4.2 μl of water was added to tube 1 (water control). 2.46 μl of pEAS002, 0.6 μl of IPTG and 1.46 μl of water was added to tube 2 (positive control).

28.3 μl of the master mix was transferred to tube 5 (A70 + RNA synthesis), along with 2 μl of each of the NTPs and 2 μl of T7 RNAP.

12 μl of A70\_MS was added to tubes 4 and 5.

20.1 μl of the mix from tube 4 was transferred to tube 3 (A70 + water), along with 2.9 μl of water. 2.9 μl of ssDNA was added to tube 4 (A70 + ssDNA)

25.1 μl of the mix from tube 5 was transferred to tube 6 (A70 + synthesis components), along with 2.9 μl of water. 2.9 μl of linearised trigger 2 DNA was added to tube 5.

The concentrations in the RNA synthesis samples were about 70 % of what they should be because our DNA concentrations were too low.

10 μl samples from each of the tubes was added to the black 384-well plate in the following order:

| 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 | 11 | 12 | 13 | 14 | 15 | 16 | 17 | 18 | 19 | 20 | 21 | 22 | 23 | 24 |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
|  | Water | A70 + water | A70 + ssDNA | A70 + synthesis components | A70 + RNA synthesis |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
|  | pos. control | A70 + water | A70 + ssDNA | A70 + synthesis components | A70 + RNA synthesis |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |

The reactions were incubated for 12 hrs, with the fluorescence at 569/593 nm was measured at 1 min intervals.

![A graph of fluorescence intensity measured in cell-free reaction as a function of time with different samples.](https://static.igem.wiki/teams/4207/wiki/220902-cell-free.png)

**Figure 54.**
mScarlet-I produced in cell-free reaction.

We failed to induce translation again.

03.09.2022 Wiki writing session

05.09.2022 Overnight cultures for some of the toeholds

Present: Jesper

**O/N cultures of A95 LacZ, A92 LacZ, B39 LacZ, Trigger 3, Zika trigger**

5 ml LB KAN50 overnight cultures of A95 LacZ, A92 LacZ, B39 LacZ, Trigger 3, Zika trigger were made.

06.09.2022 Plasmid extractions, trigger plasmid linearization, purifications and gel electrophoresis

Present: Evelina

**Plasmid extraction for overnight cultures**

Plasmid extraction with Thermo Scientific GeneJET plasmid miniprep was made according to the kit instructions and eluted to 20 µl H2O. Concentrations were measured with biodrop:

| sample | c (ng/µl) | A260/A230 | A260/A280 |
| --- | --- | --- | --- |
| Trigger 3 | 108.4 | 2.151 | 1.889 |
| Zika trigger | 73.09 | 2.025 | 1.971 |
| A92 LacZ | 32.50 | 1.182 | 1.857 |
| A95 LacZ | 64.48 | 1.593 | 1.870 |
| B39 LacZ | 38.74 | 1.632 | 1.962 |

**Trigger 1 and trigger 2 plasmid linearization**

2 reactions for trigger 1 plasmid were made. 1 µl plasmid, 5 µl rCutSmart buffer, 1 µl PstI, 1 µl H2O.

6 reactions for trigger 2 were made. 1 µl plasmid, 5 µl rCutSmart buffer, 1 µl PstI, 1 µl H2O. Incubated in 37℃ for one hour.

**Gel electrophoresis for linearised triggers.**

1% agarose gel was made. 7.5 µl of unpurified linearised trigger were mixed with 1.5 µl loading dye.

The gel was run 100 V for 35 min.

![A picture of agarose gel electrophoresis. In well 2 there is one band. Two brighter bands can be seen on well 3, with some other fainter bands.](https://static.igem.wiki/teams/4207/wiki/220906-gel.png)

**Figure 55.**
Agarose gel electrophoresis for linearised triggers 1 and 2 plasmids. Wells: 1. 5 µl ladder, 2. trigger 1, 2. trigger 2.

**Purification of linearized products**

Linearized plasmids were purified with the Monarch PCR and DNA cleanup kit, according to the manufacturer's instructions. The final elution was done with 6 µl to get the maximum concentration

07.09.2022 Team meeting + Farmer interview

Present: Anni, Iida, Jesper, Jonna, Juuli, Kristiina, Malin, Minttu, Nelli, Otto, Sofia

The main theme of our meeting was to go through our collaborations and human practices work, but we also discussed other updates.

![A picture of the Zoom window.](https://static.igem.wiki/teams/4207/wiki/aboa-notebook-meeting35.png)

07.09.2022 Cell-free reaction with A92 and A95 LacZ toeholds, ZikaV trigger plasmid linearization, purification and gel analysis

Present: Evelina, Jonna, Jesper

**Cell-free reaction with Zika-S, A92-LacZ and A95-LacZ**

The amounts of reagents were calculated using the master mix calculator, with 156 μl as the final volume. RNA synthesis was a part of each reaction, so they were added to the master mix. The following master mix was created to tube 4:

| Reagent | Volume(μl) |
| --- | --- |
| AA mix | 31.2 |
| HMP | 3.12 |
| K-G | 2.5 |
| Mg-G | 2.3 |
| Energy mix | 15.6 |
| PEG | 7.8 |
| ONPG | 1.4 |
| Each NTP solution | 2 |
| T7 RNAP | 4 |
| Extract | 38 |

The concentrations of each of the NTPs and the T7 RNAP were lower than desired, because they would've raised the volume too much. The T7 is added more than the NTPs, because there is already NTPs present in the energy mix (also there is T7 in the extract) but we reasoned this still should accelerate the synthesis.

10.1 μl of the mastermix was transferred to tube 1, along with 1.9 μl of water.

10.1 μl of the mastermix was transferred to tube 2, along with 1.1 μl pET15b-LacZ, 0.6 μl 0.02 M IPTG and 0.2 μl of water.

32.1 μl of the master mix was transferred to tubes 5 and 7.

3.9 μl of Zika-S (final concentration 5.28 nM) and 9.1 μl of water was added to tube 4.

22.6 μl from tube 4 was transferred to tube 3 with 2.4 μl of water.

0.8 μl of linearised Zika-T was added to tube 4.

13 μl of A95\_LacZ (final concentration 5.28 nM) was added to tube 5.

22.6 μl from tube 5 was transferred to tube 6 with 2.4 μl of water.

2.4 μl of linearised pODD1-Trigger1 was added to tube 4.

13 μl of A92\_LacZ (final concentration 2.6 nM) was added to tube 5. The stock concentration is so low that we couldn't get this to level with the other sensor plasmids.

22.6 μl from tube 7 was transferred to tube 8 with 2.4 μl of water.

2.4 μl of linearised pODD1-Trigger1 was added to tube 7.

The amounts of linearised triggers are lower than desired.

10 μl samples from each tube was loaded to a clear-welled 384-well plate, in the following order:

| 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 | 11 | 12 | 13 | 14 | 15 | 16 | 17 | 18 | 19 | 20 | 21 | 22 | 23 | 24 |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
|  | Water | Neg ZS | Pos ZS | Neg A95 | Pos A95 | Neg A92 | Pos A92 |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
|  | Pos control |  |  | Neg A95 | Pos A95 | Neg A92 | Pos A92 |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |

The reactions were incubated for 12 hrs at 37°C. Absorbance at 420 nm was measured at 1 min intervals.

![A graph of absorbance measured in cell-free reaction as a function of time with different samples.](https://static.igem.wiki/teams/4207/wiki/220907-cell-free.png)

**Figure 56.**
β-galactosidase activity in cell-free reaction.

**ZikaV trigger plasmid linearisation**

3 reactions were made.

1: 13.7 µl of plasmid, 5 µl rCutSmart buffer, 1 µl of AvrII, 30.3 µl H2O

2: 5.3 µl plasmid, 5 µl rCutSmart buffer, 1 µl AvrII, 38.7 µl H2O

3: approximately 5 µl plasmid, 5 µl rCutSmart buffer, 1 µl AvrII, 39 µl H2O

**ZikaV trigger linear plasmid purification**

The purification was done with NEB pcr and DNA cleanup kit according to the kit instructions. All three reactions were combined. The elution was done to 6 µl and concentration measured with biodrop.

**ZikaV linear trigger gel electrophoresis**

1 % agarose gel was made. 7.5 µl from reaction 1 was mixed with 1.5 µl loading dye and loaded to the gel. 5 µl of 1 kb ladder was added. The gel was run 100 V for 35 min.

![A picture of agarose gel electrophoresis. One bright band can be seen in the sample well.](https://static.igem.wiki/teams/4207/wiki/220907-gel.png)

**Figure 57.**
Agarose gel electrophoresis for linearised ZikaV trigger plasmid.

08.09.2022 Meeting with Aalto-Helsinki + Science Basement presentation coaching

10.09.2022 Heureka workshop + Wiki writing session

10.09.2022 Cell-free reaction with A70 and B69 mScarlet-I toeholds

Present: Jesper

**Cell-free reaction for A70\_MS & B69\_MS**

The amounts of reagents were calculated using the master mix calculator, with 156 μl as the final volume. RNA synthesis was a part of each reaction, so they were added to the master mix. The following master mix was created to tube 4:

| Reagent | Volume(μl) |
| --- | --- |
| AA mix | 31.2 |
| HMP | 3.12 |
| K-G | 2.5 |
| Mg-G | 2.3 |
| Energy mix | 15.6 |
| PEG | 7.8 |
| Each NTP solution | 2 |
| T7 RNAP | 4 |
| Extract | 38 |

As before, the RNA synthesis components are not supplied in ideal concentrations, but they seemed to work last time so we decided to replicate it.

8.65 μl of this master mix and 3.35 μl of water was transferred to tube 1 (water control).

8.65 μl of this master mix, 2 μl of pEAS002, 0.6 μl of IPTG and 0.75 μl of water was transferred to tube 1 (water control).

47.6 μl of the master mix was transferred to tube 5.

1.4 μl of A70\_MS was added to tube 4. (final concentration 6 nM)

24.5 μl of this mix and 8.5 μl of water was added to tube 3 (Neg. A70)

2.2 μl of linear Trigger 2 (final amount 244 ng) and 6.3 μl of water was added to tube 4. (Pos. A70)

14 μl of B69\_MS was added to tube 5 (final concentration 4.24 nM).

30.8 μl of this mixture and 2.2 μl of water was added to tube 6 (Neg. B69)

2.2 μl of Trigger 2 (final amount 244 ng) was added to tube 5. (Pos B69)

10 μl samples of each tube were loaded to a black 384-well plate in the following order:

| 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 | 11 | 12 | 13 | 14 | 15 | 16 | 17 | 18 | 19 | 20 | 21 | 22 | 23 | 24 |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
|  |  |  |  |  |  |  |  |  |  |  |  | Water | Neg A70 | Neg A70 | Pos A70 | Neg B69 | Neg B69 | Pos B69 |  |  |  |  |  |
|  |  |  |  |  |  |  |  |  |  |  |  | Pos. Control | Neg A70 | Pos A70 | Pos. A70 | Neg B69 | Pos B69 | Pos B69 |  |  |  |  |  |
|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |

The reactions were incubated for 12 hrs at 37°C, with the fluorescence at 569/593 nm was measured at 1 min intervals.

![A graph of fluorescence intensity measured in cell-free reaction as a function of time with different samples.](https://static.igem.wiki/teams/4207/wiki/220910-cell-free.png)

**Figure 58.**
mScarlet-I produced in cell-free reaction.

12.09.2022 Meeting with TrigGate + Meeting with Patras and TecCEM

![A picture of the Zoom view.](https://static.igem.wiki/teams/4207/wiki/aboa-notebook-220912triggate.png)

12.09.2022 Cell-free reaction with A95 and B39 LacZ toeholds, plasmid extractions, trigger plasmid linearisations and purifications

Present: Evelina, Anni

**Cell-free reaction with A95 and B39 LacZ toeholds**

The amounts of reagents were calculated using the master mix calculator, with 156 μl as the final volume. RNA synthesis was a part of each reaction, so they were added to the master mix. The following master mix was created to tube 3:

| Reagent | Volume(μl) |
| --- | --- |
| AA mix | 31.2 |
| HMP | 3.12 |
| K-G | 2.5 |
| Mg-G | 2.3 |
| Energy mix | 15.6 |
| PEG | 7.8 |
| ONPG | 1.4 |
| Each NTP solution | 2 |
| T7 RNAP | 4 |
| Extract | 38 |

10.1 μl of the mastermix was transferred to tube 1, along with 1.9 μl of water.

10.1 μl of the mastermix was transferred to tube 2, along with 1.1 μl pET15b-LacZ, 0.6 μl 0.02 M IPTG and 0.2 μl of water.

32.1 μl of the master mix was transferred to tubes 5 and 7.

3.9 μl of Zika-S (final concentration 5.28 nM) to tube 3.

16.7 μl from tube 3 was transferred to tube 4 with 2.88 μl of water.

2.4 μl of linearised Zika-T was added to tube 4.

5.28 µl of water was added to tube 3.

13 μl of B39\_LacZ (final concentration 3.4 nM) was added to tube 7.

22.6 μl from tube 7 was transferred to tube 8 with 2.4 μl of water.

2.4 μl of linearised pODD1-Trigger1 was added to tube 7.

8.8 μl of A95\_LacZ (final concentration 4 nM) was added to tube 5.

20.45 μl from tube 5 was transferred to tube 6 with 2.4 μl of water.

4.55 μl of linearised pODD1-Trigger1 was added to tube 4.

10 μl samples from each tube was loaded to a clear-welled 384-well plate, in the following order:

| 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 | 11 | 12 | 13 | 14 | 15 | 16 | 17 | 18 | 19 | 20 | 21 | 22 | 23 | 24 |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
|  |  |  |  | water | Z pos | Z neg | A95 pos | A95 neg | B39 pos | B39 neg |  |  |  |  |  |  |  |  |  |  |  |  |  |
|  |  |  |  | pos | Z pos | Z neg | A95 pos | A95 neg | B39 pos | B39 neg |  |  |  |  |  |  |  |  |  |  |  |  |  |
|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |

The reactions were incubated for 14 hrs at 37°C. Absorbance at 420 nm was measured at 1 min intervals.

![A graph of absorbance measured in cell-free reaction as a function of time with different samples.](https://static.igem.wiki/teams/4207/wiki/220912-cell-free.png)

**Figure 59.**
β-galactosidase activity in cell-free reaction.

**Plasmid extractions for pOdd-trigger 1, A95\_LZ, A92\_LZ**

Plasmid extraction with Thermo Scientific GeneJET plasmid miniprep and NEB monarch plasmid miniprep kit was made according to the kit instructions and eluted to 20/30 µl H2O. Concentrations were measured with biodrop:

| sample | c (ng/µl) | A260/A230 | A260/A280 |
| --- | --- | --- | --- |
| trigger 1 | 73.11 | 1.970 | 1.823 |
| A95 | 67.64 | 1.846 | 1.898 |
| A92 | 63.54 | 1.649 | 1.894 |

**Trigger 1 and 3 plasmid linearisation and purification**

2 reactions for each were made.

T1: 13.7 µl plasmid, 5 µl rCutSmart, 1 µl PstI, 30.3 µl H2O

T1(2): 5 µl plasmid, 5 µl rCutSmart, 1 µl PstI, 39 µl H2O

T3: 9.2 µl plasmid, 5 µl rCutSmart, 1 µl PstI, 34.8 µl H2O

T3(2): approx. 4 µl plasmid, 5 µl rCutSmart, 1 µl PstI, 40 µl H2O

Incubated at 37℃ for approximately 2.5 h.

Purification was done according to the protocol, each 2 reactions combined, elution to 6.5 µl, and concentrations were measured with biodrop:

| sample | c (ng/µl) | A260/A230 | A260/A280 |
| --- | --- | --- | --- |
| T1 | 89.83 | 1.407 | 1.840 |
| T3 | 56.70 | 1.189 | 1.909 |

13.09.2022 Farmer interview

13.09.2022 Transformation for TG B46 and TG A70 plasmids.

Present: Kristiina

14.09.2022 Team meeting

Present: Evelina, Iida, Jesper, Jonna, Kristiina, Malin, Minttu, Nelli, Otto

In this week’s team meeting, we discussed our partnerships and what’s the situation with those. In addition, we updated each other on the situation of the wiki pages and subteam activities.

![A picture of the Zoom window.](https://static.igem.wiki/teams/4207/wiki/aboa-notebook-meeting36.png)

14.09.2022 PCR for pET15b-LacZ

Present: Evelina, Kristiina, Jesper

**PCR reaction for pet15b-LacZ**

Four reactions and one water control, where DNA was replaced with MQ H2O, was made according to following:

| reagent | volume (µl) |
| --- | --- |
| Master mix | 12.5 |
| forvard primer | 1.25 |
| reverse primer | 1.25 |
| DNA | 1 |
| MQ H2O | 9 |

The following cycle was run:

| 1. | 98℃ | 1 min |  |
| --- | --- | --- | --- |
| 2. | 98℃ | 10 s |  |
| 3. | 68℃ | 30 s |  |
| 4. | 72℃ | 30 s | Steps 2-4  x 40 |
| 5. | 72℃ | 2 min |  |
| 6. | 4℃ | ∞ |  |

15.09.2022 Gel electrophoresis and extraction for PCR products, TG A70 and B46 overnight cultures

Present: Evelina, Kristiina, Jesper

**Gel electrophoresis for PCR products**

60 ml of 1 x TAE buffer was heated with 0.60 g agarose. 6 µl of SYBR safe color was added and mixed. Left to cool. Poured onto a gel tray.

Samples: 25 µl of each PCR product (4+ neg. control) was mixed with 5 µl of loading Dye and injected to the wells. Also 7 µl of ladder was injected to two wells.

![A picture of agarose gel electrophoresis. Bright bands can be seen in all of the wells around 3 kb.](https://static.igem.wiki/teams/4207/wiki/220915-gel.png)

**Figure 60.**
Agarose gel electrophoresis for LacZ PCR products

**Gel extraction**

Gel extraction was made with the NEB gel extraction kit according to the protocol. The concentrations were measured with biodrop.

**O/N miniprep cultures for TG A70 and TG B46**

Overnight cultures of TG A70 and TG B46 were made with 5 ml LB, KAN50.

16.09.2022 Meeting with Tirupati

![A picture of the Zoom view.](https://static.igem.wiki/teams/4207/wiki/aboa-notebook-220916tirupati.png)

16.09.2022 Plasmid extraction for TrigGate A70 and B46, Golden Gate assembly for both TrigGate and our own A70 and B46 toeholds and B69 toehold, transformation for assembled products

Present: Evelina, Anni

**Plasmid extraction for TG A70 and TG B46**

The extractions were made with Qiagen plasmid extraction kit according to the protocol, except the first centrifugation was 3 000 x g 5 min, and the rest centrifugations 15 500 x g. The concentrations were measured with biodrop:

| sample | c (ng/µl) | A260/A230 | A260/A280 |
| --- | --- | --- | --- |
| TG B46 | 424.3 | 2.150 | 1.875 |
| TG A70 | 423.3 | 2.248 | 1.870 |

**Golden gate assembly for TrigGateA70, TrigGateB46, A70, B46, B69**

2 reactions for each toehold (=10 reactions) were made as follows:

| Reagent | Amount | Notes | Toeholds |  |
| --- | --- | --- | --- | --- |
| pOdd1 | 1 µl |  | TG A70 | 0.24 µl |
| LacZ | 1.47 µl |  | TG B46 | 0.24 µl |
| Toehold |  |  | B69 | 0.27 µl |
| T7 terminator | 0.25 µl |  | B46 | 0.31 µl |
| T4 ligase Buffer | 2 µl |  | A70 | 0.29 µl |
| T4 ligase | 2 µl | OBS! This was supposed to be 0.25 µl |  |  |
| BsaI-HF | 0.75 µl |  |  |  |
| H2O | to 20 µl |  |  |  |

Run the following protocol with the PCR machine:

| 1. | 37℃ | 1 min |  |
| --- | --- | --- | --- |
| 2. | 16℃ | 1 min | Steps 1-2  x 30 |
| 3. | 60℃ | 5 min |  |
| 4. | 4℃ | ∞ |  |

**Transformations for golden gate products (TrigGateA70, TrigGateB46, A70, B46, B69)**

Transformations were made using heat-shock transformation protocol for *E. coli* cells. Five preps of competent DH5a cells (50 µl) were thawed on ice for 15 min. 10 µl of TrigGate A70, TrigGate B46, A70, B46 and B69 were added into separate tubes. They were incubated on ice for 30 min. Cells were heat-shocked in a 42℃ water bath for 45 s and kept on ice for 3 min. 500 µl of LB was added to each of the tubes and they were placed on 37℃, 200 rpm for 1 hr. From each of the tubes, 50 µl was plated on a LBA + Kan 50 plate. They were incubated at 37℃ overnight.

![A picture of five plates containing bacteria with five different toehold plasmids.](https://static.igem.wiki/teams/4207/wiki/220916-plates.png)

**Figure 61.**
A. Golden Gate assembled pOdd1 T7+B69+LacZ+terminator in *E.coli* DH5 α. B. Golden Gate assembled pOdd1 T7+TriGate A70+LacZ+terminator in *E.coli* DH5 α. C. Golden Gate assembled pOdd1 T7+TriGate B46+LacZ+terminator in *E.coli* DH5 α. D. Golden Gate assembled pOdd1 T7+A70+LacZ+terminator in *E.coli* DH5 α. E. Golden Gate assembled pOdd1 T7+B46+LacZ+terminator in *E.coli* DH5 α.

17.09.2022 Wiki writing session

17.09.2022 Cell-free reaction comparing autolysis and sonication batches, colony PCR for transformed toeholds

Present: Kristiina, Jesper

**Comparison of autolysis vs. sonication batches**

A master mix was created using 96 μl as the final volume. The following amounts were pipetted to tube 1:

| Reagent | Volume(μl) |
| --- | --- |
| AA mix | 19.2 |
| HMP | 1.92 |
| K-G | 1.54 |
| Mg-G | 1.41 |
| Energy mix | 9.6 |
| PEG | 4.8 |
| ONPG | 0.87 |

19.7 μl of this mix was transferred to tube 3.

16 μl of cell extract from sonication batch 2/30 (30 mg/ml) was added to tube 1.

7.8 μl of this mix was transferred to tube 2 (sonication negative) along with 4.2 μl of water.

3.3 μl of pET15b-LacZ, 1.8 μl of IPTG and 7.5 μl of water was added to tube 1 (sonication positive).

16 μl of cell extract from the autolysis batch (30 mg/ml) was added to tube 3.

7.8 μl of this mix was transferred to tube 4 (autolysis negative) along with 4.2 μl of water.

3.3 μl of pET15b-LacZ, 1.8 μl of IPTG and 7.5 μl of water was added to tube 3 (autolysis positive).

10 μl samples of each tube were loaded onto a clear-welled 384-well plate in the following order:

| 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 | 11 | 12 | 13 | 14 | 15 | 16 | 17 | 18 | 19 | 20 | 21 | 22 | 23 | 24 |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
|  | Son. neg. | Son. pos. | AL neg. | AL pos. |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
|  | Son. pos. | Son. pos. | AL pos. | AL pos. |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |

The reactions were incubated at 37°C for 3 hrs, with absorbance at 420 nm measured at 1 min intervals.

We did not get any signal.

![A graph of absorbance measured in cell-free reaction as a function of time with different samples. Colonies can be seen in all the plates.](https://static.igem.wiki/teams/4207/wiki/220917-cell-free.png)

**Figure 62.**
β-galactosidase activity in cell-free reaction.

**ColonyPCR for assembled LacZ toeholds**

ColonyPCR for TG A70, TG B46, A70, B46, B69 as follows:

| Reaction components | Volume |  | PCR program | Temperature | Time |
| --- | --- | --- | --- | --- | --- |
| Reaction volume | 250 µl |  | Initial denaturation | 98℃ | 30 s |
| Q5 High Fidelity 2x MasterMix | 125 µl |  | Cycle 40X | a) 98℃ | 10 s |
| 10 µM F Toehold Linker | 12.5 µl |  |  | b) 72℃ | 1 min |
| 10 µM R T7 Terminator | 12.5 µl |  | Final extension | 72℃ | 2 min |
| MQ Water | 100 µl |  | Hold | 4℃ | ∞ |

This 10x MasterMix was divided into 10 25 µl reactions.

Gel electrophoresis was performed.

![A picture of agarose gel electrophoresis. Faint bands and some background can be seen.](https://static.igem.wiki/teams/4207/wiki/22-09-17-colonypcr-tg46-tg70-b46-b69-a70.png)

**Figure 63.**
Agarose gel electrophoresis for colony PCR products.

19.09.2022 Colony PCR for assembled TG\_B46 and TG\_A70, PCR reaction for pET15b-LacZ and gel electrophoresis and extraction

Present: Evelina, Kristiina, Jesper

**Colony PCR for assembled TG\_B46 and TG\_A70**

The following reagents were added to a mastermix tube and divided into 5 25 µl reactions. Pipetted as follows:

| Reaction components | Volume |  | PCR program | Temperature | Time |
| --- | --- | --- | --- | --- | --- |
| Reaction volume | 125 µl |  | Initial denaturation | 98°C | 30 s |
| Q5 High Fidelity 2x MasterMix | 62.5 µl |  | Cycle 40X | a) 98°C | 10 s |
| 10 µM F Toehold Linker | 6.25 µl |  |  | b) 68°C | 1 min |
| 10 µM R T7 Terminator | 6.25 µl |  | Final extension | 72°C | 2 min |
| MQ Water | 50 µl |  | Hold | 4°C | ∞ |

All except the water control tube were inoculated with a colony from the plates. The PCR reaction was run according to the table above.

**Gel electrophoresis for PCR products**

60 ml of 1 x TAE buffer was heated with 0.60 g agarose. 6 µl of SYBR safe color was added and mixed. Left to cool. Poured onto a gel tray.

Samples: 25 µl of each PCR product was mixed with 5µl of loading Dye and injected to the wells. Also 7 µl of ladder was injected to two wells.

Run with 100 V for 35 min.

![A picture of agarose gel electrophoresis for lacZ PCR products. Faint bands can be seen in all of the wells, in the lower part of the gel (below 1 kb).](https://static.igem.wiki/teams/4207/wiki/220919-gel-colony-pcr.png)

**Figure 64.**
Agarose gel electrophoresis for colony PCR products. Wells: 1: 1 kb ladder 2: TG\_B46 (3) 3: TG\_B46 (4) 4: TG\_A70 (3) 5: TG\_A70 (4) 6: water.

**PCR reaction for pet15b-LacZ**

Four reactions and one water control, where DNA was replaced with MQ H2O, was made according to following:

| reagent | volume (µl) |
| --- | --- |
| Master mix | 12.5 |
| forvard primer | 1.25 |
| reverse primer | 1.25 |
| DNA | 1 |
| MQ H2O | 9 |

The following cycle was run:

| 1. | 98°C | 1 min |  |
| --- | --- | --- | --- |
| 2. | 98°C | 10 s |  |
| 3. | 68°C | 30 s |  |
| 4. | 72°C | 30 s | Steps 2-4  x 40 |
| 5. | 72°C | 2 min |  |
| 6. | 4°C | ∞ |  |

**Gel electrophoresis for PCR products**

60 ml of 1 x TAE buffer was heated with 0.60 g agarose. 6 µl of SYBR safe color was added and mixed. Left to cool. Poured onto a gel tray.

Samples: 25 µl of each PCR product (4+ neg. control) was mixed with 5 µl of loading Dye and injected to the wells. Also 7 µl of ladder was injected to two wells.

![A picture of agarose gel electrophoresis. Bright bands can be seen in all of the sample wells, with also some background.](https://static.igem.wiki/teams/4207/wiki/220919-gel-lacz.png)

**Figure 65.**
Agarose gel electrophoresis for LacZ PCR products.

**Gel extraction**

Gel extraction was made with the NEB gel extraction kit according to the protocol. The concentrations were measured with biodrop:

| sample | c (ng/µl) | A260/A230 | A260/A280 |
| --- | --- | --- | --- |
| 1 | 68.92 |  |  |
| 2 | 48.19 | 0.036 | 1.992 |
| 3 | 45.30 | 0.136 | 2.032 |
| 4 | 51.88 | 1.627 | 1.861 |

20.09.2022 Cell-free reaction comparing autolysis and sonication batches, Golden Gate assembly for TrigGate and our own A70 and B46 toeholds and B69 toehold, transformation for assembled products

Present: Evelina, Anni, Jesper

**Comparison of autolysis vs. sonication batches**

The reactions were incubated at 37°C for 3 hrs, with absorbance at 420 nm measured at 1 min intervals.

The samples were pipetted as follows:

| 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 | 11 | 12 | 13 | 14 | 15 | 16 | 17 | 18 | 19 | 20 | 21 | 22 | 23 | 24 |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  | Son. neg. | Son. pos. | AL neg. | AL pos. |  |
|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  | Son. pos. | Son. pos. | AL pos. | AL pos. |  |
|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |

We did not get any signal.

![A graph of absorbance measured in cell-free reaction as a function of time with different samples.](https://static.igem.wiki/teams/4207/wiki/220920-cell-free.png)

**Figure 66.**
β-galactosidase activity in cell-free reaction.

**Golden gate assembly for TrigGateA70, TrigGateB46, A70, B46, B69**

2 reactions for each toehold were made.

= 10 reactions

All except toeholds and water were pipetted to a mastermix tube, then divided into 5 tubes, then added toeholds and water and divided into 20 µl reactions. Pipetted as follows:

| Reagent | Amount | Notes | Toeholds |  |
| --- | --- | --- | --- | --- |
| pOdd1 | 1 µl |  | TG A70 | 0.24 µl |
| LacZ (from 4 different tubes) | 1.92 µl (for 5 reactions), 2.55 µl (for 3 reactions), 2.2 µl (for 1 reaction), 2.9 µl (for one reaction) |  | TG B46 | 0.24 µl |
| Toehold |  |  | B69 | 0.27 µl |
| T7 terminator | 0.25 µl |  | B46 | 0.31 µl |
| T4 ligase Buffer | 2 µl |  | A70 | 0.29 µl |
| T4 ligase | 2 µl | OBS! This was supposed to be 0.25 µl |  |  |
| BsaI-HF | 0.75 µl |  |  |  |
| H2O | to 20 µl |  |  |  |

Run the following protocol with the PCR machine:

| 1. | 37°C | 1 min |  |
| --- | --- | --- | --- |
| 2. | 16°C | 1 min | Steps 1-2  x 30 |
| 3. | 60°C | 5 min |  |
| 4. | 4°C | ∞ |  |

**Transformations for golden gate products (TrigGateA70, TrigGateB46, A70, B46, B69)**

Transformations were made using heat-shock transformation protocol for *E. coli* cells. Five preps of competent DH5a cells (50 µl) were thawed on ice for 15 min. 10 µl of TrigGate A70, TrigGate B46, A70, B46 and B69 were added into separate tubes. They were incubated on ice for 30 min. Cells were heat-shocked in a 42°C water bath for 45 s and kept on ice for 3 min. 500 µl of LB was added to each of the tubes and they were placed on 37°C, 200 rpm for 1 hr. From each of the tubes, 50 µl was plated on a LBA + Kan 50 plate. They were incubated at 37 °C overnight.

![A picture of five plates containing bacteria with five different toehold plasmids.](https://static.igem.wiki/teams/4207/wiki/220920-plates.png)

**Figure 67.**
A. Golden Gate assembled pOdd1 T7+TriGate B46+LacZ+terminator in *E.coli* DH5 α. B. Golden Gate assembled pOdd1 T7+B69+LacZ+terminator in *E.coli* DH5 α. C. Golden Gate assembled pOdd1 T7+B46+LacZ+terminator in *E.coli* DH5 α. D. Golden Gate assembled pOdd1 T7+TriGate A70+LacZ+terminator in *E.coli* DH5 α. E. Golden Gate assembled pOdd1 T7+A70+LacZ+terminator in *E.coli* DH5 α.

21.09.2022 Team meeting

Present: Juuli, Malin, Minttu, Nelli

We discussed our Education work and how to proceed with it. Another theme for this meeting was to finally conclude which special prizes we are aiming to achieve and who will be responsible for those wiki pages.

![A picture of the Zoom window.](https://static.igem.wiki/teams/4207/wiki/aboa-notebook-meeting37.png)

21.09.2022 Colony PCR twice for assembled plasmids

Present: Evelina, Anni, Kristiina, Jesper

**Colony PCR for assembled LacZ toeholds**

Colony PCR for TG A70, TG B46, A70, B46, B69, 2 reactions for each plus a water control as follows:

| Reaction components | Volume |  | PCR program | Temperature | Time |
| --- | --- | --- | --- | --- | --- |
| Reaction volume |  |  | Initial denaturation | 98°C | 30 s |
| Q5 High Fidelity 2x MasterMix | 137.5 µl |  | Cycle 35X | a) 98°C | 10 s |
| 10 µM F Toehold Linker | 13.75 µl |  |  | b) 72°C | 1 min |
| 10 µM R T7 Terminator | 13.75 µl |  | Final extension | 72°C | 2 min |
| MQ Water | 110 µl |  | Hold | 4°C | ∞ |

This 10x MasterMix was divided into 11 25 µl reactions. All except the water control tube were inoculated with a colony from the plates. The PCR reaction was run according to the table above.

**Gel electrophoresis for PCR products**

60 ml of 1 x TAE buffer was heated with 0.60 g agarose. 6 µl of SYBR safe color was added and mixed. Left to cool. Poured onto a gel tray.

Samples: 25 µl of each PCR product (4+ neg. control) was mixed with 5 µl of loading Dye and injected to the wells. Also 7 µl of ladder was injected to two wells.

![A picture of agarose gel electrophoresis. Faint bands can be seen in all wells.](https://static.igem.wiki/teams/4207/wiki/220921-gel-1.png)

**Figure 68.**
Agarose gel electrophoresis for colony PCR products. Wells: 1: 1 kb ladder, 2:TG B46 1, 3:TG B46 2, 4:B46 1, 5:B46 2, 6:A70 1, 7:A70 2, 8: 1 kb ladder, 9:B69 1, 10:B69 2, 11:TG B46 1, 12:TG B46 2, 13:H2O

**ColonyPCR for assembled LacZ toeholds**

ColonyPCR for TG A70, TG B46, A70, B46, B69, 2 reactions for each. We also included PCR reactions for purified A95-LacZ-plasmid and a colony PCR reaction for it. Reactions as follows:

| Reaction components | Volume |  | PCR program | Temperature | Time |
| --- | --- | --- | --- | --- | --- |
| Reaction volume |  |  | Initial denaturation | 98°C | 30 s |
| Q5 High Fidelity 2x MasterMix | 137.5 µl |  | Cycle 35X | a) 98°C | 10 s |
| 10 µM F Toehold Linker | 13.75 µl |  |  | b) 72°C | 1 min |
| 10 µM R T7 Terminator | 13.75 µl |  | Final extension | 72°C | 2 min |
| MQ Water | 110 µl |  | Hold | 4°C | ∞ |

This 10x MasterMix was divided into 13 25 µl reactions. All except the water control tube were inoculated with a colony from the plates. The PCR reaction was run according to the table above.

We also started mini cultures for the colonies marked 3 from all the plates.

The electrophoresis was run at 100 V for 40 minutes.

![A combined figure containing two separate pictures of separate agarose gel electrophoresis experiments for colony PCR products.  Four bands can be seen from the upper picture. Two bands can be seen from the lower picture.](https://static.igem.wiki/teams/4207/wiki/220921-gel-2.png)

**Figure 69.**
Agarose gel electrophoresis for colony PCR products. A. Upper row of the gel: TG 46-3, TG46-¤, TG 70-3, Ladder, TG 70-4, B69-3, B69-4, Ladder, B46-3, B46-4. B. Lower row of the gel: A70-3, A70-4, A95-colony, Ladder, A95 purified plasmid, water control.

We can see that the PCR was successful for the previously confirmed A95-LacZ. We can also see faint bands in TG46-3 and A70-4. We'll do PCR for purified plasmids tomorrow.

22.09.2022 Plasmid extractions and two PCR reactions for them, trigger plasmid linearisations

Present: Evelina, Anni, Kristiina, Jesper

**Plasmid extraction for TG A70, TG B46, A70, A92, A95, B39, B46, B69, T1, T2, T3, ZT and pET15b-LacZ**

The extractions were made with Qiagen plasmid extraction kit according to the protocol, except the first centrifugation was 3 000 x g 5 min, and the rest centrifugations 15 500 x g. The concentrations were measured with Biodrop:

| plasmid | ng/µl | A260/A230 | A260/A280 |
| --- | --- | --- | --- |
| T1 | 99.19 | 1.319 | 1.901 |
| T2 | 335.0 | 2.204 | 1.872 |
| T3 | 73.77 | 1.277 | 1.855 |
| ZT | 40.00 | 1.333 | 1.905 |
| TG 70 | 310.8 | 2.103 | 1.863 |
| TG 46 | 259.5 | 2.068 | 1.860 |
| A70 | 337.7 | 2.050 | 1.869 |
| A92 | 105.7 | 1.539 | 1.896 |
| A95 | 73.73 | 1.578 | 1.904 |
| B39 | 60.88 | 1.489 | 1.910 |
| B46 | 240.0 | 2.106 | 1.890 |
| B69 | 230.8 | 2.102 | 1.879 |
| pET15b-LacZ | 66.17 | 1.689 | 1.937 |

**PCR x2 for extracted plasmids TG 70, TG 46, A70, B46, B69 and A95 as a positive control**

Two reactions were made of each plasmid, one with F Toehold Linker and T7 terminator primers, and another with LacZ primers. The following 7x master mixes were made and divided into 25 µl reactions that were added 1 µl of plasmid DNA.

| Mastermix1 | Volume |  | Mastermix2 marked with blue dot | Volume |
| --- | --- | --- | --- | --- |
| Q5 HF 2X MM | 87.5 µl |  | Q5 HF 2X MM | 87.5 µl |
| F toehold linker | 8.75 µl |  | F LacZ | 8.75 µl |
| R T7 terminator | 8.75 µl |  | R LacZ | 8.75 µl |
| MQ H2O | 70 µl |  | MQ H2O | 70 µl |

The PCR program:

| PCR program | Temperature | Time |
| --- | --- | --- |
| Initial denaturation | 98°C | 30 s |
| Cycle 35X | a) 98°C | 10 s |
|  | b) 72°C | 1 min |
| Final extension | 72°C | 2 min |
| Hold | 4°C | ∞ |

**Plasmid linearization for T1, T2, T3 and ZT**

The following reactions were incubated at 37℃ for 4 h: 5 µl rCutSmart, 1 µl PstI, 1 µg plasmid DNA (10 µl T1, 3 µl T2, 14 µl T3, 25 µl ZT), and MQ water to 50 µl.

23.09.2022 Cell-free reaction comparing autolysis and sonication batches, linearized trigger purifications and RNA synthesis for them, gel electrophoresis for PCR products and linearized triggers

Present: Evelina, Anni, Kristiina, Jesper

**Comparison of autolysis vs. sonication batches**

We repeated the previous cell-free measurements, we took a new amino acid mix and energy mix. Samples were pipetted in the following order:

| 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 | 11 | 12 | 13 | 14 | 15 | 16 | 17 | 18 | 19 | 20 | 21 | 22 | 23 | 24 |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  | Son. neg | Son. pos | AL neg | AL pos |  |
|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  | Son. pos | Son. neg | AL pos | AL pos |  |
|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |

![A graph of absorbance measured in cell-free reaction as a function of time with different samples.](https://static.igem.wiki/teams/4207/wiki/220923-cell-free.png)

**Figure 70.**
β-galactosidase activity in cell-free reaction.

**Gel electrophoresis for PCR products and linearized triggers**

120 ml of 1 x TAE buffer was heated with 1.20 g agarose. 12 µl of SYBR safe color was added and mixed. Left to cool. Poured onto a gel tray.

Samples: 25 µl of each PCR product (4+ neg. control) was mixed with 5 µl of loading Dye and injected to the wells. 1,5 µl loading dye was added to 7,5 µl linear plasmids. Also 7 µl of 1 kb ladder was added. The gel was run at 100 V for 35 min.

![A picture of agarose gel electrophoresis. Figure A shows one clear band for sample wells 3-6, with another faint band on the upper part of the gel. In well two only some background is seen. Well 7 is empty, well 8 shows a few faint bands. In figure B, very clear bands are shown for wells 2-7, they also show another faint band at the bottom of the gel. In well 8 some faint bands are shown, one that matches the size of the lower band in the sample wells.](https://static.igem.wiki/teams/4207/wiki/220923-gel.png)

**Figure 71.**
Agarose gel electrophoresis for PCR products and linearised trigger plasmids. A. Upper row of the gel (F toehold linker and T7 terminator primers): 1: Ladder, 2: TG B46, 3: TG A70, 4: B69, 5: B46, 6: A70, 7: A95, 8: H2O, 9: Ladder, 10: Trigger 1, 11: Trigger 2, 12: Trigger 3, 13: ZikaV trigger. B. Lower row of the gel (LacZ primers): 2: Ladder, 3: TG B46, 4: TG A70, 5: B69, 6: B46, 7: A70, 8:A95, 9: H2O, 10: Ladder.

**Purification for linearized triggers**

The purification was made with NEB Monarch PCR & DNA cleanup kit according to the protocol. The concentrations were measured with BioDrop:

| sample | c (ng/µl) | A260/A230 | A260/A280 |
| --- | --- | --- | --- |
| trigger 1 | 14.76 | 1.073 | 1.903 |
| trigger 2 | 43.64 | 1.638 | 1.846 |
| trigger 3 | 14.60 | 1.000 | 1.697 |
| Zika trigger | 15.77 | 1.340 | 1.798 |

**RNA synthesis for T1 T2 T3 ZT**

Four 20 µl RNA synthesis reactions were made according to the RNA protocol: 1.5 µl of all of the following reagents NTPs (ATP, CTP, GTP, UTP), T7 RNA Polymerase, and T7 RNA Polymerase reaction Buffer (all from NEB) and 11 µl linearised plasmid (T1 T2 T3 or ZT). The reactions were incubated at 37°C. overnight.

24.09.2022 Science Basement talk + Wiki writing session

24.09.2022 RNA purification for triggers

Present: Jonna, Kristiina

**RNA purification for T1 T2 T3 ZT**

T1 T2 T3 ZT RNA was purified according to the Monarch RNA miniprep Kit (NEB) and the concentrations were measured with Biodrop:

|  | ng/µl | A260/A230 | A260/280 |
| --- | --- | --- | --- |
| T1 | 252.4 | 2.400 | 1.9 |
| T2 | 168.7 | 2.347 | 1.869 |
| T3 | 367.7 | 2.037 | 1.711 |
| ZT | 167.8 | 2.311 | 1.944 |

26.09.2022 Meeting with Patras and TecCEM

![A picture of the Zoomm view.](https://static.igem.wiki/teams/4207/wiki/aboa-notebook-220926patras.png)

26.09.2022 Cell-free reaction comparing autolysis and sonication batches, plasmid extraction for pET15b-LacZ

Present: Evelina, Kristiina, Jesper

**Comparison of autolysis vs. sonication batches**

A master mix was created using 144 μl as the final volume. The following amounts were pipetted to tube 1:

| Reagent | Volume(μl) |
| --- | --- |
| AA mix | 28.8 |
| HMP | 2.88 |
| K-G | 2.3 |
| Mg-G | 2.13 |
| Energy mix | 14.4 |
| PEG | 7.2 |
| ONPG | 1.3 |

19.7 μl of this mix was transferred to tubes 3 and 5.

16 μl of cell extract from sonication batch 2/30 (30 mg/ml) was added to tube 1.

7.8 μl of this mix was transferred to tube 2 (sonication negative) along with 4.2 μl of water.

10.7 μl of pET15b-LacZ, 1.8 μl of IPTG and was added to tube 1 (sonication positive).

16 μl of cell extract from the autolysis batch (30 mg/ml) was added to tube 3.

7.8 μl of this mix was transferred to tube 4 (autolysis negative) along with 4.2 μl of water.

10.7 μl of pET15b-LacZ, 1.8 μl of IPTG and was added to tube 3 (autolysis positive).

16 μl of cell extract from sonication batch 41 was added to tube 5.

7.8 μl of this mix was transferred to tube 6 (sonication 41 negative) along with 4.2 μl of water.

10.7 μl of pET15b-LacZ, 1.8 μl of IPTG and was added to tube 5.

10 μl samples of each tube were loaded onto a clear-welled 384-well plate in the following order:

| 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 | 11 | 12 | 13 | 14 | 15 | 16 | 17 | 18 | 19 | 20 | 21 | 22 | 23 | 24 |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
|  |  |  |  |  |  |  |  |  |  |  | AL neg | AL pos | 31 neg | 31 pos | 41 neg | 41 pos |  |  |  |  |  |  |  |
|  |  |  |  |  |  |  |  |  |  |  | AL pos | AL pos | 31 pos | 31 pos | 41 pos | 41 pos |  |  |  |  |  |  |  |
|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |

The reactions were incubated at 37°C for 3 hrs, with absorbance at 420 nm measured at 1 min intervals.

![A graph of absorbance measured in cell-free reaction as a function of time with different samples.](https://static.igem.wiki/teams/4207/wiki/220926-cell-free.png)

**Figure 72.**
β-galactosidase activity in cell-free reaction.

**Plasmid extraction for pET15b-LacZ**

The extractions were made with Qiagen plasmid extraction kit according to the protocol, except the first centrifugation was 5 000 x g 5 min, and the rest centrifugations 15 000 x g. The concentrations were measured with Biodrop.

Concentration 21.91 ng/ul

A260/A280 1.840

A260/A230 1.470

27.09.2022 Cell-free reaction with PURExpress, phenol-chloroform extractions for toehold plasmids

Present: Evelina, Kristiina, Jesper

**Cell-free reaction with PURExpress**

The following reagents were pipetted to tubes:

| reagent | water control | pos control | Zika pos | Zika neg |
| --- | --- | --- | --- | --- |
| Solution A | 4 µl | 4 µl | 4 µl | 4 µl |
| Solution B | 3 µl | 3 µl | 3 µl | 3 µl |
| ONPG | 0.2 µl | 0.2 µl | 0.2 µl | 0.2 µl |
| IPTG |  | 0.6 µl |  |  |
| pET15b-LacZ |  | 2.5 µl |  |  |
| ZikaV sensor |  |  | 2.8 µl | 2.8 µl |
| Trigger RNA |  |  | 1.2 µl |  |
| water | 3 µl |  |  | 1.2 µl |
| volume | 10.2 µl | 10.2 µl | 10.2 µl | 10.2 µl |

9.5 μl samples of each tube were loaded onto a clear-welled 384-well plate in the following order:

| 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 | 11 | 12 | 13 | 14 | 15 | 16 | 17 | 18 | 19 | 20 | 21 | 22 | 23 | 24 |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  | water |  |
|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  | pos |  |
|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  | Zika pos |  |
|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  | Zika neg |  |
|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |

The reactions were incubated at 37°C for 6 hrs, with absorbance at 420 nm measured at 1 min intervals.

![A graph of absorbance measured in cell-free reaction as a function of time with different samples.](https://static.igem.wiki/teams/4207/wiki/220927-cell-free.png)

**Figure 73.**
β-galactosidase activity in cell-free reaction.

**Phenol chloroform extraction for toeholds**

Previously extracted plasmids were diluted to 200 µl. 200 µl of phenol-chloroform was added and mixed for 5 min, then spinned 12 000 x g for 5 min. Approximately 180 µl of the top phase was pipetted to a new tube and 180 µl chloroform was added and mixed for 1 min, then spinned 12 000 x g for 5 min. Approximately 150 µl of the top phase was pipetted to a new tube and 375 µl of 100 % ethanol and 15 µl of Na-Ac buffer (pH 5.2) was added and left to room temperature for 15 min, the spinned at 14 000 x g for 10 min. The pellet was washed with 1 ml 70 % ethanol and spinned at 14 000 x g for 10 min. The supernatant was discarded and then the tubes were spinned for 15 s and the rest of the ethanol was pipetted away. The pellets were left to dry for approximately 5 min and diluted to 25 µl H2O. The concentrations were measured with biodrop:

| sample | c (ng/µl) | A260/A230 | A260/A280 |
| --- | --- | --- | --- |
| B46 | 27.42 | 2.043 | 1.778 |
| TG B46 | 66.84 | 2.167 | 1.721 |
| A70 | 74.21 | 2.049 | 1.758 |
| A92 | 5.725 | 1.212 | 2.101 |
| TG A70 | 56.60 | 2.128 | 1.736 |
| ZS | 42.76 | 2.279 | 1.965 |

28.09.2022 Team meeting

Present: Anni, Evelina, Iida, Jesper, Jonna, Juuli, Kristiina, Malin, Minttu, Nelli, Otto

In this week’s meeting, we mainly discussed the situation with different wiki texts and things that we still need to do regarding it. In addition, we updated each other about recent progress with collaborations, partnerships, and dry lab work.

28.09.2022 Cell-free reaction with PURExpress, plasmid extraction and phenol-chloroform extraction for B46 and A70 toeholds

Present: Evelina, Anni, Kristiina, Jesper

**Cell-free reaction with PURExpress**

We repeated the last measurement, this time making sure we vortex the solution A well.

| 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 | 11 | 12 | 13 | 14 | 15 | 16 | 17 | 18 | 19 | 20 | 21 | 22 | 23 | 24 |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  | water |  |  |
|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  | pos |  |  |
|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  | Zika pos |  |  |
|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  | Zika neg |  |  |
|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |

**Plasmid extraction for B46 and A70**

Plasmid extraction was done with Qiagen plasmid miniprep kit according to the protocol.

**Phenol chloroform extraction for toeholds B46 and A70**

Previously extracted plasmids were diluted to 250 µl. 250 µl of phenol-chloroform was added and mixed for 5 min, then spinned 12 000 x g for 5 min. Approximately 220 µl of the top phase was pipetted to a new tube and 220 µl chloroform was added and mixed for 1 min, then spinned 12 000 x g for 5 min. Approximately 200 µl of the top phase was pipetted to a new tube and 500 µl of 100 % ethanol and 20 µl of Na-Ac buffer (pH 5.2) was added and left to room temperature for 15 min, then spinned at 14 000 x g for 10 min. The pellet was washed with 1 ml 70 % ethanol and spinned at 14 000 x g for 10 min. The supernatant was discarded and then the tubes were spinned for 15 s and the rest of the ethanol was pipetted away. The pellets were left to dry for approximately 5 min and diluted to 25 µl H2O. The concentrations were measured with Biodrop:

| sample | c (ng/µl) | A260/A230 | A260/A280 |
| --- | --- | --- | --- |
| B46 | 192.7 | 2.330 | 1.806 |
| A70 | 67.58 | 2.285 | 1.707 |

30.09.2022 Meeting with TrigGate

30.09.2022 Cell-free reaction with our own system, ssDNA and RNA synthesis

Present: Jesper

**Cell-free with our own system, ssDNA and RNA synthesis**

The following master mix was created:

| Reagent | Volume(μl) |
| --- | --- |
| AA mix | 32 |
| HMP | 3.2 |
| K-G | 2.56 |
| Mg-G | 2.36 |
| Energy mix | 16 |
| PEG | 8 |
| Extract | 39 |

15.5 μl was transferred to a tube labeled along with 0.6 μl of X-GAL 9. 7.95 μl of this mixture was transferred to tube 10, along with 4.05 μl of water (X-GAL neg).

2.95 of pET15b-LZ was added to tube 4, alo9ng with 0.6 μl of IPTG and 0.45 μl of water (X-GAL pos).

43.8 μl of the remaining master mix was transferred to tube 5, along with 0.87 μl of ONPG. 0.88 μl of each NTP and 1.76 μl of T7 RNAP was added.

8.8 μl of this mixture was transferred to tube 7, along with 3.2 μl of water (RNA synth. water).

8.8 μl was transferred to tube 8 along with 2.6 μl pET15b and 0.6 μl of IPTG (RNA synth. control).

16.2 μl of ZS was added to tube 5.

24.2 μl of this mixture was transferred to tube 6, along with 1 μl of water (synthesis neg).

1 μl of linear ZT-plasmid was added to tube 5 (synthesis pos.).

0.87 μl of ONPG was added to tube 4.

7.89 μl of this mix was transferred to tube 1, along with 4.11 μl of water (ssDNA water).

7.89 μl was transferred to tube 2, along with 2.6 μl pET15b, 0.6 μl of IPTG and 0.9 μl of water (ssDNA control).

16.2 μl of ZS was added to tube 4.

22.6 μl of this mixture was transferred to tube 3, along with 1.1 μl of water (ssDNA neg.).

1.1 μl of ZT ssDNA was added to tube 4 (ssDNA pos).

The reaction was incubated and measured as before.

Samples pipetted as follows:

| 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 | 11 | 12 | 13 | 14 | 15 | 16 | 17 | 18 | 19 | 20 | 21 | 22 | 23 | 24 |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
|  |  |  |  |  | 9 | 7 | 3 | 6 |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
|  |  |  |  |  | 10 | 8 | 3 | 6 |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
|  |  |  |  |  | 1 |  | 4 | 5 |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
|  |  |  |  |  | 2 |  | 4 | 5 |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |

![A graph of absorbance measured in cell-free reaction as a function of time with different samples.](https://static.igem.wiki/teams/4207/wiki/220930-cell-free-420.png)

**Figure 74.**
β-galactosidase activity in cell-free reaction. Measured with absorbance at 420 nm.

![A graph of absorbance measured in cell-free reaction as a function of time with different samples.](https://static.igem.wiki/teams/4207/wiki/220930-cell-free-615.png)

**Figure 75.**
β-galactosidase activity in cell-free reaction. Measured with absorbance at 615 nm.

OCT

01.10.2022 Wiki writing session

03.10.2022 Cell-free reaction with PURExpress

Present: Jesper

**Cell-free reaction with PURExpress**

The following reagents were pipetted to tubes:

| reagent | 1(water control) | 2(DHFR) | 3(DHFR + T7) | 4(pET15b+ONPG in water) | 5(pET15b+ONPG in DMSO) | 6 (pEAS002) | 7(pEAS002 +T7) |
| --- | --- | --- | --- | --- | --- | --- | --- |
| Solution A | 4 µl | 4 µl | 4 µl | 4 µl | 4 µl | 4 µl | 4 µl |
| Solution B | 3 µl | 3 µl | 3 µl | 3 µl | 3 µl | 3 µl | 3 µl |
| ONPG |  |  |  | 0.4 µl | 0.4 µl |  |  |
| T7 RNAP | 1 |  | 1 | 1 | 1 |  | 1 |
| IPTG | 0 | 0 | 0 | 0.6 | 0.7 | 0.8 | 0.9 |
| DNA | 0 | 0.8 | 0.8 | 1 | 1 | 1.4 | 1.4 |
| water | 3 µl | 2.2 | 1.2 | 1.2 µl |  |  |  |
| volume | 10.2 µl | 10.2 µl | 10.2 µl | 10.2 µl |  |  |  |

9.5 μl samples of each tube were loaded onto a clear-welled 384-well plate in the following order:

| 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 | 11 | 12 | 13 | 14 | 15 | 16 | 17 | 18 | 19 | 20 | 21 | 22 | 23 | 24 |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  | 1 | 5 |  |  |  |  |  |  |  |
|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  | 2 | 6 |  |  |  |  |  |  |  |
|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  | 3 | 7 |  |  |  |  |  |  |  |
|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  | 4 |  |  |  |  |  |  |  |  |
|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |

The reactions were incubated at 37°C for 6 hrs, with absorbance at 420 nm measured at 1 min intervals.

05.10.2022 Media interview

05.10.2022 Team meeting

Present: Anni, Evelina, Iida, Jesper, Jonna, Juuli, Kevät, Kristiina, Malin, Minttu, Nelli, Otto

We recapped the situation with different wiki texts.

05.10.2022 Cell-free reaction with our own system, ssDNA and RNA synthesis, phenol-chloroform extraction

Present: Evelina, Jesper

**Cell-free reaction with PURExpress**

The following reagents were pipetted to tubes:

| reagent | 1(water control) | 2(DHFR) | 3(DHFR + T7) | 4(pET15b+ONPG in water) | 5(pET15b+ONPG in DMSO) | 6 (pEAS002) | 7(pEAS002 +T7) |
| --- | --- | --- | --- | --- | --- | --- | --- |
| Solution A | 4 µl | 4 µl | 4 µl | 4 µl | 4 µl | 4 µl | 4 µl |
| Solution B | 3 µl | 3 µl | 3 µl | 3 µl | 3 µl | 3 µl | 3 µl |
| ONPG |  |  |  | 0.4 µl | 0.4 µl |  |  |
| T7 RNAP | 1 |  | 1 | 1 | 1 |  | 1 |
| IPTG | 0 | 0 | 0 | 0.6 | .7 | 0.8 | 0.9 |
| DNA | 0 | 0.8 | 0.8 | 1 | 1 | 1.4 | 1.4 |
| water | 3 µl | 2.2 | 1.2 | 1.2 µl |  |  |  |
| volume | 10.2 µl | 10.2 µl | 10.2 µl | 10.2 µl |  |  |  |

9.5 μl samples of each tube were loaded onto a clear-welled 384-well plate in the following order:

| 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 | 11 | 12 | 13 | 14 | 15 | 16 | 17 | 18 | 19 | 20 | 21 | 22 | 23 | 24 |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  | 1 | 5 |  |  |  |  |  |  |  |
|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  | 2 | 6 |  |  |  |  |  |  |  |
|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  | 3 | 7 |  |  |  |  |  |  |  |
|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  | 4 |  |  |  |  |  |  |  |  |
|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |

The reactions were incubated at 37°C for 6 hrs, with absorbance at 420 nm measured at 1 min intervals.

**Cell-free with our own system, ssDNA and RNA synthesis**

The following master mix was created:

| Reagent | Volume(μl) |
| --- | --- |
| AA mix | 32 |
| HMP | 3.2 |
| K-G | 2.56 |
| Mg-G | 2.36 |
| Energy mix | 16 |
| PEG | 8 |
| ONPG | 0.88 |
| Extract | 39 |

7.8 μl was transferred to tube 1 along with 4.2 μl of water.

7.8 μl was transferred to tube 2, along with .,1 μl of pET15b-LacZ, 1 μl IPTG and 2.1 μl water (pos. control).

9.5 μl of ZS was added to the mastermix.

19.3 μl was transferred to tube 3, along with 1.1 μl of ssDNA and 1.7 μl of water (ssDNA pos).

2.8 μl was added to tube 4 (ssDNA neg.).

The reaction was incubated and measured as before.

Samples pipetted as follows:

| 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 | 11 | 12 | 13 | 14 | 15 | 16 | 17 | 18 | 19 | 20 | 21 | 22 | 23 | 24 |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
|  |  |  |  |  |  |  |  |  | 1 | 3 |  |  |  |  |  |  |  |  |  |  |  |  |  |
|  |  |  |  |  |  |  |  |  | 2 | 3 |  |  |  |  |  |  |  |  |  |  |  |  |  |
|  |  |  |  |  |  |  |  |  | 4 |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
|  |  |  |  |  |  |  |  |  | 4 |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |

![A graph of absorbance measured in cell-free reaction as a function of time with different samples.](https://static.igem.wiki/teams/4207/wiki/221005-cell-free.png)

**Figure 76.**
β-galactosidase activity in cell-free reaction. Measured with absorbance at 420 nm.

**Phenol chloroform extraction for Zika V sensor (day1)**

Previously extracted plasmids were diluted to 250 µl. 250 µl of phenol-chloroform was added and mixed for 5 min, then spinned 12 000 x g for 5 min. Approximately 230 µl of the top phase was pipetted to a new tube and 230 µl chloroform was added and mixed for 1 min, then spinned 12 000 x g for 5 min. Approximately 210 µl of the top phase was pipetted to a new tube and 525 µl of 100 % ethanol and 21 µl of Na-Ac buffer was added and left to -20℃  o/n.

06.10.2022 School visits

06.10.2022 Cell-free reaction with PURExpress, cell-free reaction with A70, B46, TG\_A70 and TG\_B46 and ssDNA triggers, phenol-chloroform extraction

Present: Jesper

**Cell-free reaction with PURExpress**

The following reagents were pipetted to tube 2 (Zika neg):

| reagent | volume (µl) |
| --- | --- |
| Solution A | 40 |
| Solutioon B | 30 |
| ONPG | 1.5 |

14 µl was transferred to tube 1 (water), 6 µl water was added.

24 µl ZikaV sensor was added to tube 2 and 40 µl was transferred to tube 3 (zika pos) with 5 µl ZikaV trigger RNA. 5 µl water was added to tube 2.

20 µl samples were pipetted as follows:

| 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 | 11 | 12 | 13 | 14 | 15 | 16 | 17 | 18 | 19 | 20 | 21 | 22 | 23 | 24 |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  | 3 | 1 |  |  |  |  |
|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  | 3 | 2 |  |  |  |  |
|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  | 2 |  |  |  |  |
|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |

The reactions were incubated 3 h at 37°C and absorbance measured at 420 nm at 1 min intervals.

![A graph of absorbance measured in cell-free reaction as a function of time with different samples.](https://static.igem.wiki/teams/4207/wiki/221006-cell-free-pure.png)

**Figure 77.**
β-galactosidase activity in cell-free reaction. Measured with absorbance at 420 nm.

**Cell-free reaction with A70, B46, TG70 and TG46 and ssDNA triggers**

The following mastermix was created to tube 3 (A70 neg):

| Reagent | Volume (µl) |
| --- | --- |
| AA mix | 40 |
| HMP | 4 |
| K-G | 3.2 |
| Mg-G | 2.96 |
| Energy mix | 20 |
| PEG | 10 |
| ONPG | 3 |
| Extract | 48.8 |

7.8 µl mastermix was transferred to tube 1 (water control), 4.2 µl of water was added.

7.8 µl mastermix was transferred to tube 2 (pos control), 1.1 µl pET15b-LacZ, 1 µl IPTG and 2.1 µl water was added.

29 µl mastermix was transferred to tube 5 (B46 neg), 16 µl of B46 was added and 22.5 µl transferred to tube 6 (B46 pos) with 1.1 µl ssDNA. 1.1 µl water was added to tube 5.

29 µl mastermix was transferred to tube 7 (TG70 neg), 7.9 µl of TG70 was added and 18.45 µl transferred to tube 8 (TG70 pos) with 1.1 µl ssDNA and 2.45 µl water. 5.5 µl water was added to tube 7.

29 µl mastermix was transferred to tube 9 (TG46 neg), 6.7 µl of TG70 was added and 17.85 µl transferred to tube 10 (TG70 pos) with 1.1 µl ssDNA and 1.45 µl water. 2.55 µl water was added to tube 9.

6 µl of A70 was added to tube 3 and 17.5 µl transferred to tube 4 (A70 pos) with 1.1 µl ssDNA and 3.4 µl water. 4.5 µl water was added to tube 3.

10 µl samples was pipetted as follows:

| 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 | 11 | 12 | 13 | 14 | 15 | 16 | 17 | 18 | 19 | 20 | 21 | 22 | 23 | 24 |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
|  |  |  |  |  |  |  |  |  |  |  | 3 | 5 | 7 | 9 |  |  |  |  |  |  |  |  |  |
|  |  |  |  |  |  |  |  |  |  |  | 3 | 5 | 7 | 9 |  |  |  |  |  |  |  |  |  |
|  |  |  |  |  |  |  |  |  |  | 1 | 4 | 6 | 8 | 10 |  |  |  |  |  |  |  |  |  |
|  |  |  |  |  |  |  |  |  |  | 2 | 4 | 6 | 8 | 10 |  |  |  |  |  |  |  |  |  |
|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |

The reactions were incubated 3 h at 37°C and absorbance measured at 420 nm at 1 min intervals.

![A graph of absorbance measured in cell-free reaction as a function of time with different samples.](https://static.igem.wiki/teams/4207/wiki/221006-cell-free-ssdna.png)

**Figure 78.**
β-galactosidase activity in cell-free reaction. Measured with absorbance at 420 nm.

**Phenol chloroform extraction for Zika V sensor (day 2)**

The tube was spinned at 14 000 x g for 10 min. The pellet was washed with 1 ml 70 % ethanol and spinned at 14 000 x g for 10 min. The supernatant was discarded and then the tubes were spinned for 15 s and the rest of the ethanol was pipetted away. The pellets were left to dry for approximately 5 min and diluted to 25 µl H2O. The concentration was measured with biodrop: 39,38 ng/µl.

07.10.2022 Cell-free reaction with Zika, A70, TG\_A70 and TG\_B46, plasmid extraction, phenol-chloroform extraction

Present: Evelina, Kristiina, Jesper

**Cell-free reaction with Zika, A70, TG70 and TG46**

The following mastermix was created to tube 3 (Zika neg):

| Reagent | Volume (µl) |
| --- | --- |
| AA mix | 40 |
| HMP | 4 |
| K-G | 3.2 |
| Mg-G | 2.96 |
| Energy mix | 20 |
| PEG | 10 |
| ONPG | 3 |
| Extract | 48.8 |

7.8 µl mastermix was transferred to tube 1 (water control), 4.2 µl of water was added.

7.8 µl mastermix was transferred to tube 2 (pos control), 1.1 µl pET15b-LacZ, 1 µl IPTG and 2.1 µl water was added.

29 µl mastermix was transferred to tube 5 (A70 neg), 6 µl of A70 was added and 17.5 µl transferred to tube 6 (A70 pos) with 3.6 µl trigger 2 RNA and 0.9 µl water. 4.5 µl water was added to tube 5.

29 µl mastermix was transferred to tube 7 (TG70 neg), 7.9 µl of TG70 was added and 18.45 µl transferred to tube 8 (TG70 pos) with 3.6 µl trigger 2 RNA. 3.6 µl water was added to tube 7.

29 µl mastermix was transferred to tube 9 (TG46 neg), 6.7 µl of TG70 was added and 17.85 µl transferred to tube 10 (TG70 pos) with 2.2 µl trigger 3 RNA and 1.95 µl water. 4.15 µl water was added to tube 9.

16.94 µl ZikaV sensor was added to tube 3 and 22.97 µl transferred to tube 4 (Zika pos pos) with 2.7 µl ZikaV trigger RNA. 2.7 µl water was added to tube 3.

10 µl samples was pipetted as follows:

| 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 | 11 | 12 | 13 | 14 | 15 | 16 | 17 | 18 | 19 | 20 | 21 | 22 | 23 | 24 |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  | 7 | 9 |  |
|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  | 7 | 9 |  |
|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  | 8 | 10 |  |
|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  | 8 | 10 |  |
|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  | 1 | 3 | 5 |  |  |  |  |  |
|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  | 2 | 3 | 5 |  |  |  |  |  |
|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  | 4 | 6 |  |  |  |  |  |
|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  | 4 | 6 |  |  |  |  |  |
|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |

The reactions were incubated 3 h at 37°C and absorbance measured at 420 nm at 1 min intervals.

![A graph of absorbance measured in cell-free reaction as a function of time with different samples.](https://static.igem.wiki/teams/4207/wiki/221007-cell-free.png)

**Figure 79.**
β-galactosidase activity in cell-free reaction. Measured with absorbance at 420 nm.

**Plasmid extraction for zikaV sensor**

The extraction was done with Qiagen plasmid miniprep kit according to the protocol.

**Phenol cloroform extraction for Zika V sensor**

The extraction was done according to our protocol, the concentration was measured with biodrop (31.09 ng/µl).

08.10.2022 Wiki writing session

{% endblock %}

{% extends "layout.html" %}
{% block title %}Engineering{% endblock %}
{% block lead %}{% endblock %}
{% block title\_class %}engineering-title{% endblock %}
{% block page\_content %}

* [Cycle 0](#cycle0)
* [Cycle 1](#cycle1)
* [Cycle 2](#cycle2)
* [Cycle 3](#cycle3)
* [Cycle 4](#cycle4)
* [Cycle 5](#cycle5)
* [Cycle 6](#cycle6)
* [Cycle 7](#cycle7)
* [Cycle 8](#cycle8)
* [References](#references)

Plant diseases are an increasing threat to global food security. As plant infections can rarely be visualized at early phases of plant-pathogen encounter, we sought to devise a bioassay kit that could identify an infection before phenotypic symptoms arise. For this purpose we developed a low-cost cell-free system to be used on-site, based on exchangeable toehold switches that respond to pathogen-specific gene sequences. After thorough research we got inspired by the toehold publications by Arce et al (2021) and Pardee et al (2016) and partially based our project on those. As a demonstration, we built a novel toehold switch for detecting barley yellow dwarf virus (BYDV), and compiled a library of alternative switches towards other pathogens to showcase system modularity.

As the ultimate demonstration of our engineering success we managed to design and build a working cell-free expression system that activates when a pathogen-specific trigger sequence is present in the reaction. We also confirmed the modularity of the detection kit and identified subsequent steps in the process that are necessary to develop the system towards commercialization. There is still work to do and therefore future iGEM teams can continue brainstorming and developing our modular system.

The project was subdivided into a number of consecutive design-build-test-learn (DBTL) cycles, as presented in a simplified sequence below. The entire process included seven completed interconnected iterations (Cycles 0-6), and two uncompleted cycles, which represent the next steps of the future work.

![A representative illustration of the DBTL-process. The engineering design cycle consists of four arrows in a circle representing design, build, test and learn.](https://static.igem.wiki/teams/4207/wiki/engineering-success-dbtl-tehty-biorender.png)

## Cycle 0: Design of novel toehold switches

---

**Design**

Novel toehold switches were designed using design principles, shown to produce viable toehold switches. In an effort to detect the target pathogen, we designed several toehold switches that were outlined at the sites of conserved regions of pathogen genomes. More about the design of the toehold switches is presented on our [design page](https://2022.igem.wiki/aboa/design).

In our project, we wanted to create a novel system by using a single target pathogen, which we decided to be the barley yellow dwarf virus (BYDV). We partnered with the iGEM teams TAU and IISER-Tirupati for designing more efficient toeholds for the virus. The partnerships were essential when designing our system. Along with BYDV, we designed toehold switch sequences for other target pathogens, such as the tomato brown rugose fruit virus (TBRFV) and the potato virus Y (PVY). Total of 12 pathogens including BYDV were compiled into a library of alternative switches. Rest of the pathogens can be found on the [design page](https://2022.igem.wiki/aboa/design). In order to exemplify the modularity, we also partnered with iGEM teams Patras and TecCEM, gathering information about other possible detectable pathogens. This partnership with Patras and TecCEM was an intrinsic part of building the pathogen library, thus successfully demonstrating the global impact of our project. More about the partnership can be found on our [partnership page](https://2022.igem.wiki/aboa/partnership).

**Build**

Toehold switches were generated using an original design algorithm. The algorithm can be accessed on our [GitLab page](https://gitlab.igem.org/2022/software-tools/aboa/-/tree/main/).

**Test**

The performance of the designed toehold switches was estimated using computational modeling.

**Learn**

Based on the modeling, the best performing BYDV toehold switches were selected for laboratory testing. This cycle was performed multiple times, more information can be seen on [Design](https://2022.igem.wiki/aboa/design), [Results](https://2022.igem.wiki/aboa/results) and [Model](https://2022.igem.wiki/aboa/model).

## Cycle 1: Verification of the construct assembly system

---

**Design**

We selected Golden Gate cloning as the assembly platform for generating the toehold sensor plasmids (see [Design](https://2022.igem.wiki/aboa/design)). We designed our sensor plasmids so that the toehold switch is easily exchangeable with Golden Gate assembly.

**Build**

The reporter gene fragments for β-galactosidase (β-gal) and mScarlet-I were generated by PCR, while the rest of the parts were ordered as synthetic fragments from IDT. Components arrived in IDT Golden Gate compatible kanamycin A plasmids and we transformed them into the DH5α-cells. T7 promoter and T7 terminator were ordered from IDT as ready fragments, whereas the toehold switch sequences were self-constructed. The plasmid backbone, pOdd1 (BBa\_J428381), was provided in the iGEM distribution kit. The constructs were assembled using Golden Gate cloning, and transformed into the *E. coli* DH5α strain.

The modularity of the detection kit is based on the standardized junction sites used in Golden Gate assembly. These junction sites correspond to the iGEM Type IIS standards, with slight modifications. Using this approach, we produced several variants of our sensor plasmid using toehold switches and different reporters. More information can be seen on our [Design page](https://2022.igem.wiki/aboa/design).

**Test**

Clone candidates harboring the successfully assembled constructs were selected based on kanamycin resistance and the color of the colonies on the transformant plates, white indicating a plasmid carrying an insert fragment. The presence of the construct with a correct-sized insert, indicating successful Golden Gate assembly, was further verified using colony PCR.

**Learn**

The Golden Gate assembly was successful based on the acquired fragment sizes, and allowed the isolation of all fourteen toehold sensor plasmids used in the successive analysis (Cycle 6). This also demonstrated the modularity of the assembly strategy, which allows the pathogen-specific toehold sequences in the reporter plasmid to be flexibly replaced for the identification of other plant pathogens of interest. More information can be found on the [Results page](https://2022.igem.wiki/aboa/results).

## Cycle 2: Confirmation of the β-gal reporter system / signal detection

---

**Design**

To confirm the functionality of the reporter system and the detection method used in the work, we set to analyze cells expressing β-gal in a well plate format.

**Build**

*E. coli* BL21 (DE3) strain harboring the plasmid construct for ꞵ-gal expression was acquired from our university.

**Test**

The β-gal strain was cultured in the presence of IPTG (isopropyl β-D-1-thiogalactopyranoside) and colorimetric substrate (ONPG, 2-Nitrophenyl β-D-galactopyranoside) to induce expression, and analyzed spectrophotometrically at 420 nm using a plate reader.

**Learn**

The observed signal in the induced cells confirmed that the β-gal detection system was functional, and that the well plate system could be applied for the analysis for the toehold constructs. More information can be found on the [Results page](https://2022.igem.wiki/aboa/results).

## Cycle 3: Confirming the cell-free expression system functionality

---

**Design**

We decided to use crude cell extracts obtained from the lysis of *E. coli* BL-21-Gold-dLac (DE3) as the basis of the cell-free system (see [Design](https://2022.igem.wiki/aboa/design)). The lysates were to be tested first with the reporter, which was shown to be functional in the previous Cycle 2.

**Build**

The cells were grown, lysed and used in the construction of a cell-free expression (CFE) system. The CFE system was combined with the rest of the components required for the detection, including the β-gal expression plasmid, IPTG and ONPG substrate (see [Experiments](https://2022.igem.wiki/aboa/experiments)).

**Test**

The β-gal signal was measured spectrophotometrically the same way as in the previous *in vivo* assay (Cycle 2).

**Learn**

The observed β-gal signal confirmed that the crude lysate CFE system was functional, and could be applied in the successive cycles in the work (Cycles 4-6). However, the signal from the system decreased over time, suggesting the CFE system should be further optimized (Cycle 8). More information can be seen on the [Results page](https://2022.igem.wiki/aboa/results).

## Cycle 4: Comparison of alternative lysis methods for preparing the CFE system

---

**Design**

Next we evaluated two different lysis methods to optimize the CFE system tested in Cycle 3 (see [Design](https://2022.igem.wiki/aboa/design)).

**Build**

*E. coli* BL-21-Gold-dLac (DE3) cells were cultured and lysed using either a probe sonicator or a phage λ-based autolysis system. The crude cell extracts from two different lysing methods were used in the construction of two distinct CFE systems and combined with expression plasmids for β-gal and mScarlet-I (see [Experiments](https://2022.igem.wiki/aboa/experiments)) .

**Test**

The acquired parallel lysates were tested for the expression of two alternative reporters, β-gal (Cycles 2 and 3) and mScarlet-I.

**Learn**

Both lysis systems were functional. The test set-up revealed differences between the methods; sonication appeared to be more effective for both β-gal and mScarlet-I. More information can be found on our [Results page](https://2022.igem.wiki/aboa/results).

## Cycle 5: Verification of the function of a control toehold system

---

**Design**

Next, the CFE system generated by sonication in Cycle 4 was to be tested in context with a known functional toehold system with β-gal as the reporter (Pardee et al, 2016)(Cycle 3-5). For this, we found suitable toehold switch plasmid that had previously been shown to work in a cell-free context (see [Design](https://2022.igem.wiki/aboa/design)).

**Build**

The analyzed toehold construct (ZIKV\_Sensor\_27B\_LacZ) was a gift from James Collins & Alexander Green, acquired from Addgene. The plasmid was amplified and extracted from *E. coli* DH5α.

**Test**

The toehold construct was used for setting up a CFE reaction evaluated in previous Cycles 3 and 4. The system was analyzed with and without the toehold-specific trigger for the expression of ꞵ-gal reporter spectrophotometrically at 420 nm using a plate reader. The trigger was provided as either ssDNA or RNA sequences in parallel reactions.

**Learn**

The analyzed toehold reporter system was shown to be functional in the developed CFE system. Importantly, the measured ꞵ-gal signal was induced in the presence of the trigger, with a lower nonspecific background signal in its absence. However, the activity of the toehold sensor could not be measured with RNA triggers, necessitating further testing and optimization of the CFE system (Cycle 8). More information can be found on the [results page](https://2022.igem.wiki/aboa/results).

## Cycle 6: Analysis of the assembled toehold constructs in the CFE system

---

**Design**

Next, we wanted to test the BYDV-specific toehold constructs assembled in Cycle 1 in the established CFE system (Cycle 5) (see [Design](https://2022.igem.wiki/aboa/design)).

**Build**

Four of the 14 assembled plasmids (Cycle 1) were amplified and extracted, and in order to use them for parallel CFE reactions as in the previous successful test reaction (Cycle 5).

**Test**

The parallel reactions were monitored for ꞵ-gal signal (spectrophotometrically as in previous Cycle 5) in the presence and absence of the toehold-specific triggers, against negative and positive controls (see [Results](https://2022.igem.wiki/aboa/results)).

**Learn**

One of the tested BYDV toehold sensor plasmids was clearly functional and responded to the presence of the specific trigger (i.e. specific BYDV DNA fragment), as seen in the increase of the ꞵ-gal signal as compared to the negative control. The observed trigger-speficity was less pronounced than in the case of the control toehold tested in Cycle 5. The other three tested BYDV toehold systems did not produce the desired β-gal response in the presence of the trigger sequences in our reaction setup. Further testing is necessary for evaluating the applicability of the toeholds and for system optimization, as discussed in Cycle 8.

## Cycle 7: Analysis of the remaining toeholds

---

**Design**

To obtain conclusive results on the functionality of the designed BYDV sensors, additional testing is required. This will include activity screens for the remaining untested BYDV toeholds, as well as appropriate repetitions of the trials in Cycle 6.

**Build / Test / Learn**

To be done.

## Cycle 8: Optimization of the CFE system

---

**Design**

Once all the functional BYDV toeholds have been identified, the reaction needs to be optimized for sensitivity and maximal signal levels. This will include evaluation of the CFE reaction conditions, as well as additional fine-tuning of the toeholds at sequence level, before the system can be optimized for the bioassay format.

**Build / Test / Learn**

To be done.

## References

---

* Arce, A., Guzman Chavez, F., Gandini, C., Puig, J., Matute, T., Haseloff, J., Dalchau, N., Molloy, J., Pardee, K., & Federici, F. (2021). Decentralizing Cell-Free RNA Sensing With the Use of Low-Cost Cell Extracts. *Frontiers in Bioengineering and Biotechnology*, 9, 744. https://doi.org/10.3389/FBIOE.2021.727584/BIBTEX

* Pardee, K., Green, A. A., Takahashi, M. K., Braff, D., Lambert, G., Lee, J. W., Ferrante, T., Ma, D., Donghia, N., Fan, M., Daringer, N. M., Bosch, I., Dudley, D. M., O’Connor, D. H., Gehrke, L., & Collins, J. J. (2016). Rapid, Low-Cost Detection of Zika Virus Using Programmable Biomolecular Components. *Cell*, 165(5), 1255–1266. https://doi.org/10.1016/J.CELL.2016.04.059

{% endblock %}

{% extends "layout.html" %}
{% block title %}Experiments{% endblock %}
{% block lead %}{% endblock %}
{% block title\_class %}experiments-title{% endblock %}
{% block page\_content %}

* [General experiments](#general_experiments)
* [Lysate production](#lysate_production)
* [The PURE system](#PURE)
* [Golden Gate assembly](#golden_gate)
* [Cell-free reactions](#cell_free)
* [References](#references)

# General experiments / Toolbox

---

### Heat-shock transformation for *E. coli* cells

Thaw the competent DH5a/BL21 cells on ice for 15 min. Add 1 ul of desired plasmid, for example, purified pEAS001 (mScarlet), into the tube. Incubate the cells on ice for 30 min. Heat-shock the cells in a 42°C water bath for 45 seconds and keep on ice for 5 min. Add 500 ul of LB into the tube. Place the culture on 37°C, 200-250 rpm for 1 hour. Plate 100 ul from the tube on an LBA plate containing desired antibiotic and the remaining 400 ul on a second plate. The volumes can be varied depending on the cell culture’s growth. Incubate at 37°C.

### PCR

PCR reactions were conducted with Q5 High-Fidelity 2 X MasterMix (NewEngland BioLabs, United States) (NewEngland BioLabs, n.d.) according to the manufacturer’s protocol. This protocol was varied to suit our needs by testing different temperatures, times and cycles.

**Table 1.** PCR reaction conditions.

|  |  |  |
| --- | --- | --- |
| 1. Initial Denaturation | 98°C | 30 s |
| 2. 25-35 cycles | 98°C  50-72°C  72°C | 5-10 s  10-30 s   20-30 s/kb |
| 3. Final Extension | 72°C | 2 min |
| 4. Hold | 4°C | ∞ |

**Table 2.** Primers used in reporter gene amplifications and colony PCRs.

| Primers | Sequence | Tm (°C) |
| --- | --- | --- |
| lacZ forward | GGTCTCTAATGACCATGATTACGGATTCACTGGCC | 59.46 |
| lacZ reverse | GGTCTCTAACCTTATTTTTGACACCAGACCAACTGGTAATGG | 58.81 |
| mScarlet forward | GGTCTCTAATGGTTAGCAAGGGTGAGGCAGTG | 59.28 |
| mScarlet reverse | GGTCTCTAAGCTCACTTATAAAGCTCGTCCATGCCG | 59.62 |
| mScarlet-i forward | GGTCTCTAATGGTTAGCAAGGGTGAGGCAGTG | 59.28 |
| mScarlet-i reverse | GGTCTCTAAGCTCACTTATAAAGCTCGTCCATGCCG | 59.62 |

### Preparation of plates

Melt 100 ml of LBA. Add 100 µl of the desired antibiotic (either kanamycin 50 mg/ml, ampicillin 100 mg/ml or tetracycline 10-15 mg/ml) to the melted LBA. Mix and pour onto the plates. Leave to solidify. Apply parafilm and store the plates in a refrigerator at +4°C.

### Preparation of growth media

Prepare the growth media according to the tables and autoclave them.

**Table 3.** 2 x YT per 1 liter

|  |  |
| --- | --- |
| Tryptone | 16 g |
| Yeast extract | 10 g |
| Sodium chloride | 5 g |

**Table 4.** 4 x YTP+P per 1 liter

|  |  |
| --- | --- |
| Tryptone | 16 g |
| Yeast extract | 10 g |
| Sodium chloride | 5 g |
| Potassium phosphate monobasic | 3.99 g |
| Potassium phosphate dibasic | 6.97 g |

### Competent cells

**Day 1)**

Inoculate the cells in 5 ml of LB and grow overnight.

**Day 2)**

From the overnight culture, 0.1 - 0.5 % inoculate into 100 ml of LB and grow the cells to OD600= ~0.4-0.5. Incubate the culture at 4°C for 30 minutes. Centrifuge the cells at 5000rpm for 5 mins at 4°C in 50-ml Falcon tubes. Discard the supernatant and suspend the cells in each tube in 25 ml (or 50% of the original volume) of ice-cold sterile 0.1M MgCl2. Incubate on ice for 15 mins. Centrifuge the cells at 5000rpm at 4°C for 5 mins. Discard the supernatant and resuspend them in 25 ml of ice-cold sterile 0.1M CaCl2 and incubate in ice for 15 mins. Centrifuge the cells again (5000rpm for 5 mins at 4°C) and discard the supernatant. Resuspend the cells in each tube in 1 ml of ice-cold sterile 0.1M CaCl2 supplemented with 15-20% glycerol and aliquote 100 µl into each 1.5 ml tube. Store immediately at -80°C.

### Plasmid extraction

Plasmid extraction was conducted with the Monarch® Plasmid Miniprep Kit (New England Biolabs, United States) according to the manufacturer’s protocol.

### Digestion

Plasmids, for example ZikaV Sensor and ZikaV Trigger, were digested according to the following protocol. EcoRI HF was used for ZikaV Sensor and AvrII was used for Zika V Trigger.

**Table 5.** Digestion reaction.

| Reaction component | Concentration in the reaction |
| --- | --- |
| DNA | 1 µg |
| rCutSmart | 1 X |
| Restriction enzyme | 1 U |
| H2O | Up to reaction volume |

### Agarose gel electrophoresis

Prepare 1 % agarose gel by dissolving 0.60 g of agarose into 60 ml of 1 x TAE Buffer in the microwave and adding 6 µl of SYBR Safe (Thermofisher, United States) into the mixture. Pour the gel onto the gel tray and let it solidify.

### Gel extraction

Gel extraction was conducted with the Monarch® DNA Gel Extraction Kit (New England Biolabs, United States) according to the manufacturer’s protocol.

### Liquid cultures

Kanamycin (50 µg/ml) was used for toehold plasmids B69, B46 and B39, reporter plasmids pEAS001 and pEAS002 and for the control plasmid, ZikaV-Sensor. Ampicillin (100 µg/ml) was used for the reporter plasmid pET15b-LacZ, the control plasmid ZikaV-Trigger and the autolysis plasmid pAD-LyseR. Tetracycline (10 µg/ml) was used for BL21-Gold-deLac.

To prepare minipreps for strains containing desired plasmids, pick a single colony from a plate and inoculate it in 5 ml of 2xYT and desired antibiotic. Grow overnight at 200-250 rpm at 37°C.

# Lysate production

---

Lysates provide the protein synthesis machinery necessary for the transcription of the toehold switches and the translation of reporter genes that become activated when the toehold switch recognizes its trigger sequence. In our project, we compared the effect of two distinct cell lysis methods, the sonication and the crude cell preparation autolysis, on the performance of the cell-free detection system. Autolysis is an innovative method described by Didovyk et al. (2017), in which the cell kills itself to produce the cell lysate. For autolysis, you only need to transform the autolysis plasmid into the desired cell strain and freeze the cells at -80°C to induce cell death. Lysis by sonication is based on sound waves that create cell membrane disrupting bubbles (ThermoFisher Scientific, n.d.) . The comparison gives us the opportunity to make our system as effective and optimal as possible. In addition, by using these approaches, our system will have more impact as an easy and affordable detection system as we can diminish the costs by using easier and cheaper production methods.

### Autolysis by freeze-thawing

This autolysis protocol is based on work by Didovyk, Tonooka, Tsimring & Hasty, 2017.

**Materials needed:**

- LB plates supplemented with antibiotics

- LB/2xYT media supplemented with antibiotics

- 2xYTPG media supplemented with antibiotics

- S30A buffer: 50 mM Tris-HCl (pH 7.7), 60 mM potassium glutamate, 14 mM magnesium glutamate. Final pH adjusted to 7.7

- S30A+DTT: S30A supplemented with 2mM DTT

- *E. Coli* BL21-Gold-dLac

- 1 L flask

**Growing and harvesting cells**

Grow the cells with autolysis plasmid overnight on LBA + ampicillin plates at 37℃. Start the starter cultures by picking single colonies from the plates and growing them overnight at 37℃ in 4 ml of 2xYT+ampicillin medium. Start the production cultures by inoculating 400 µl of starter culture in 400 ml of 2 x YTPG + ampicillin medium and growing the cultures at 37℃ on a shaker (300 rpm) in a 1 L flask until optical density (OD600: 600 nm wavelength, 1 cm path) was reached. Harvest the cells by centrifugation for 15 mins, 1800 g at room temperature, when the 5-fold culture dilution had OD600 of 0.3.

Discard the supernatant and resuspend the pellet in circa 45 ml of cold (4-10℃) S30A buffer by vortex mixing. Transfer the resuspension to a pre-weighed 50 ml tube and centrifuge as earlier (15 min, 18000 g). Remove the supernatant and weigh the pellet. Resuspend the pellet in two volumes (pellet to buffer ratio 1:2) of cold S30A+DTT. For example, 2.6 ml of buffer was added to 1.3 g pellet.

A direct quote containing a note from the work by Didovyk et al, 2017: “Optimization: the quality and quantity of autolysate are sensitive to the pellet to buffer ratio; more concentrated autolysates usually have higher transcription-translation activity, however high concentration of cells leads to high viscosity of the solution after lysis and the lysate becomes difficult to clean of debris. The ratio can be optimized within the range of 1:1.8 - 1:2.5.”

Vortex and freeze the mix in -80℃ for 15 minutes.

A direct quote containing a note from the work by Didovyk et al, 2017: “Freezing cells in liquid nitrogen could potentially increase the quality.”

**Freeze-thawing autolysates**

Thaw the frozen cell suspensions in a water bath at room temperature and vortex vigorously for 2-3 min and incubate at 37℃ on a shaker at 300 rpm for 45 min. Vortex the mix one more time and incubate as above (45 min, 37℃, 300 rpm).

Note: Reducing the duration of runoff reaction (thawing), now 90 minutes, could potentially improve the performance when using *E. Coli* B strain.

Clear the sample by centrifugation for 45-60 min at 30 000 g at 4℃.

Note: Optionally longer centrifugation at 21 000 g, however higher speed helps to compact the sample.

Pipette the supernatant out carefully and centrifuge briefly (3-5 min, 21 000 g).

Optional step: The last centrifugation step can be repeated if the lysate is not largely free of cell debris.

Split the lysate into small aliquots, for example 50 µl or 100 µl, in Eppendorf tubes and freeze at -80℃ or use for cell or used for cell-free expression directly.

### Sonication

Materials needed for sonication:

- Ice

- Cells on ice

- 200 ml plastic decanter tube (might be available at the sonication place)

**Table 6.** Sonication cycle.

|  |  |
| --- | --- |
| 1. Sonication | 30 s |
| 2. Rest | 30-40 s |
| 4 x Cycle |  |

Maintain the cells on ice all the time. Remember to submerge the sonicator tip well but make sure that it doesn’t touch the tube anywhere. Please use hearing protection when running the machine.

We used Soniprep150 (MSE, the USA) for sonication.

# The PURE system

---

The PURE system is a cell-free protein expression system that is constructed from individual components of protein synthesis, such as polymerases and ribosomes. It consists of four subsystems: transcription, aminoacylation, translation and energy regeneration. (Matsubayashi & Ueda, 2014) Like lysates, it provides the necessary protein synthesis machinery for the transcription of the toehold switches and the translation of activated reporter genes.

PURE Protein Synthesis was conducted with PURExpress® In Vitro Protein Synthesis Kit (New England BioLabs) according to the manufacturer’s protocol, except we did not use the RNAse inhibitor. Instead, phenol-chloroform extraction was used to purify plasmid preps and to avoid RNA degradation.

### Phenol-chloroform extraction (1:1)

**Phenol-chloroform extraction**

Add 1 x phenol:chloroform (V = 1:1) solution to the sample. Mix gently for 5 minutes by turning the tube a few times (do not vortex). Centrifuge for 5 minutes at 12 000 x g and transfer the top phase into a new tube for chloroform extraction.

Add 1 x chloroform extraction (V = 1:1) solution to the sample. Mix gently for 5 minutes by turning the tube a few times (do not vortex). Centrifuge for 5 minutes at 12 000 x g and transfer the top phase into a new tube for chloroform extraction.

**Precipitating DNA**

Add 2.5 x final volume of cold absolute ethanol, let precipitate in -80℃ for 1-2 hours. Alternatively precipitate the DNA at room temperature by adding Na-Ac (pH 5.0-5.3), 0.1 x of final volume and 2.5 x final volume absolute ethanol.

Collect the precipitate by centrifuging at 14 000 x g at 4°C for 10 min. Wash the precipitate by adding 1 ml of cold 70 % ethanol, vortexing and centrifuging as above. Remove the wash solution, spin the tubes and remove the rest of the wash solution by pipetting. Let the tubes dry with caps open.

Dilute the precipitation in 25 µl of MQ water, TE buffer or Tris buffer (1-10 mM TrisHCl pH 8).

# Golden Gate assembly of toehold switches and sensors

---

Golden Gate assembly is a fast single-pot cloning method that enables the digestion and the subsequent ligation of multiple inserts simultaneously and isothermally. This is enabled by the clever design of cleavage sites and the use of IIS restriction enzymes. (Siddiqui, Sharma & Yazdani 2022) You can read more about Golden Gate assembly on Dry lab webpage. With the Golden Gate assembly, we were able to assemble our toehold switches and sensors fast and reliably.

### Golden Gate Assembly of the toehold switch and trigger plasmids

Golden Gate Assembly was conducted with NEBridge® Golden Gate Assembly Kit (BsaI-HF® v2) (New England BioLabs) according to the manufacturer’s protocol.

# Cell-free reaction and absorbance/fluorescence assay

---

Cell-free systems provide the necessary transcriptional and translational machinery for production without living cells that can spread and mutate. Cell lysates and the PURE system, a mix reconstituted from purified *E. coli* components, provide the necessary proteins and ribosomes for protein translation. (Garenne et al., 2021) In our cell free reactions, we combine either lysate or the PURE system, energy mix, amino acid mix, buffers, our trigger RNA and DNA containing the reporter gene.

Absorbance and fluorescence assays are where our reporter genes, mScarlet, mScarlet-i and lacZ come into play. If the toehold switch recognizes its trigger sequence obtained from BYDV, the detection system produces either fluorescence or a color detectable either with our plate reader or with our eyes.

### RNA synthesis of the trigger sequence

Thaw the necessary kit components, mix and pulse-spin in microfuge to collect solutions to the bottom of tubes. Keep on ice. Add the reagents in the following order to the reaction tube: MQ water up to 20 µl, 1.5 µl 10 X RNAP reaction buffer (NEB); 1.5 µl each of the NTPs: ATP (100 mM), GTP (100 mM), CTP (100 mM), UTP (100 mM); 1 µg of linearized DNA and 1.5 µl T7 RNAP (NEB). 20 µl reaction. Incubate the reaction at 37°C for 4-16 hours. This protocol was modified from the Protocol for Standard RNA Synthesis by NEB.

**Table 7.** RNA synthesis reaction.

| Reagent | Stock concentration | Concentration in the reaction (V = 20 µl) |
| --- | --- | --- |
| 10 X RNAP reaction buffer | 10 X | 1 X |
| ATP | 100 mM | 7.5 mM |
| GTP | 100 mM | 7.5 mM |
| CTP | 100 mM | 7.5 mM |
| UTP | 100 mM | 7.5 mM |
| Linearised template DNA | - | 1 µg |
| T7 RNAP | 50 000 U/ml | 75 U |

### RNA purification

RNA purification was conducted with the Monarch® Total RNA Miniprep Kit (New England Biolabs, United States) according to the manufacturer’s protocol.

### Reagents for cell-free reaction

**Table 8.** Energy mix.

| Components | Stock concentration | Concentration in the energy mix |
| --- | --- | --- |
| HEPES | 2 M | 0.51 M |
| ATP + GTP | 75 nM / 56 nM | 15.11 nM / 11.28 nM |
| GTP | 100 nM | 3.71 nM |
| CTP | 100 nM | 9.00 nM |
| UTP | 100 nM | 9.00 nM |
| tRNA | 50 mg/ml | 2.00 nM |
| CoA | 65 mM | 2.60 mM |
| NAD | 175 mM | 2.02 mM |
| Folinic acid | 33.9 M | 0.68 M |
| Spermidine | 1 M | 0.01 M |
| Maltodextrin | 650 mg/ml | 121.64 mg/ml |

**Table 9.** Amino acid mix.

| Component | Concentration in the reaction |
| --- | --- |
| Leucine | 5 mM |
| Alanine, arginine, asparagine, aspartic acid, glutamine, glutamic acid, glycine, histidine, isoleucine, lysine, methionine, phenylalanine, proline, serine, threonine, valine, tryptophan, tyrosine, cysteine | 6 mM |

### Cell-free reaction

**Table 10.** Cell-free reaction MasterMix. These concentrations depend on previous steps, such as plasmid or RNA purification, and need to be calculated separately each time.

| Component | Concentration in the reaction | Volume (µl), V = 10 µl |
| --- | --- | --- |
| Amino Acid mix (X) | 1 | 2 |
| HMP (mg/ml) | 0.6 | 0.2 |
| K-G (µM) | 56 | 0.16 |
| Mg-G (µM) | 1.48 | 0.148 |
| ONPG (mM) | 0.3 | 1 |
| PEG (%) | 2 | 0.5 |
| DTT (1 M) | - | 0.129 |
| Cell extract (mg/ml) | 10 | 2.44 |
| DNA (nM) | 3 | 3.85 |
| Trigger RNA (nM) | 500 | 1.23 |

Make three cell-free reaction samples with at least three replicates: 1) one water control without the toehold switch plasmid nor the trigger sequence, 2) one negative control without the trigger sequence and 3) one with both the toehold switch plasmid and the trigger sequence. You can prepare a MasterMix for each sample (Table 10). When preparing samples 1) and 2), add MQ-water instead of DNA and/or trigger RNA.

Load a desired volume, for example 5-20 µl, to a plate. You can also test different volumes for the same sample in the same run. We used a clear V-bottom 96-plate. Incubate the reaction at a desired temperature for a desired amount of time. We used various temperatures and times. 37°C and three hours worked the best for us.

Note: The concentrations of the reagents and the parameters of the run should be optimized to suit your needs. The concentrations and parameters we used might not work for you in the best possible way.

# References

---

* Didovyk, A., Tonooka, T., Tsimring, L., & Hasty, J. (2017). Rapid and Scalable Preparation of Bacterial Lysates for Cell-Free Gene Expression. *ACS synthetic biology*, 6(12), 2198–2208. https://doi.org/10.1021/acssynbio.7b00253

* Garenne, D., Haines, M.C., Romantseva, E. F.,Freemont, P., Strychalski, E.A. & Noireaux, V. (2021). Cell-free gene expression. *Nat Rev Methods Primers* 1, 49. https://doi.org/10.1038/s43586-021-00046-x

* Matsubayashi, H., & Ueda, T. (2014). Purified cell-free systems as standard parts for synthetic biology. *Current opinion in chemical biology*, 22, 158–162. https://doi.org/10.1016/j.cbpa.2014.09.031

* NewEngland Biolabs. (n.d.) NEBridge® Golden Gate Assembly Kit (BsaI-HF® v2). Retrieved October 1, 2022 from https://www.neb.com/products/e1601-nebridge-golden-gate-assembly-mix-basai-hf-v2

* NewEngland Biolabs. (n.d.) Protocol for Q5® High-Fidelity 2X Master Mix. Retrieved October 1, 2022 from https://international.neb.com/protocols/2012/12/07/protocol-for-q5-high-fidelity-2x-master-mix-m0492

* New England BioLabs. (n.d.) Protocol for Standard RNA Synthesis. Retrieved October 7, 2022 from https://international.neb.com/protocols/2015/03/09/protocol-for-standard-rna-synthesis

* Siddiqui, M. A., Sharma, A. & Yazdani, S. S. (2022) Design, building, and challenges in synthetic genomics. In Singh, V (Ed.), *New frontiers and applications of synthetic biology* (Singh, Ed.), (pp. 71). Academic Press.

* ThermoFisher Scientific. (n.d.) Traditional Methods of Cell Lysis for Protein Extraction. Retrieved October 1, 2022 from https://www.thermofisher.com/fi/en/home/life-science/protein-biology/protein-biology-learning-center/protein-biology-resource-library/pierce-protein-methods/traditional-methods-cell-lysis.html#:~:text=Sonication%20of%20cells%20is%20the,spores%2C%20and%20finely%20diced%20tissue.

{% endblock %}

{% extends "layout.html" %}
{% block title %}Results{% endblock %}
{% block lead %}{% endblock %}
{% block title\_class %}results-title{% endblock %}
{% block page\_content %}

* [Design of novel toehold switches](#design)
* [Successful Golden Gate assembly](#golden_gate)
* [Validating the measurement technique](#measurement)
* [Demonstration of CFE viability](#cfe)
* [Confirming toehold functionality in sCFE system](#toehold)
* [Discussion](#discussion)
* [References](#references)

We sought to create a cell-free diagnostic kit for the detection of plant pathogens. Our kit uses toehold-switch based sensing of the pathogen's genome to facilitate the detection. The translation of a reporter protein is activated only in the presence of a conserved sequence in a pathogen’s genome.

Our system consists of two major components: a cell-free expression system and a sensor plasmid, which contains the toehold switch regulating the translation of the reporter protein. In our wet lab efforts, we successfully produced a viable cell-free expression system and assembled the sensor plasmids. We successfully combined the functionalities of these major components, although further testing and optimization would be needed. We also showcased our system capable of toehold switch-based sensing and managed to prove the functionality of a novel sensor plasmid. We produced a promising system, which could be transformed into a functional detection kit with further optimization.

## Design of novel toehold switches

---

First we started by aligning ten barley yellow dwarf virus whole genome entries from GenBank. These aligned genomes were screened for at least 36-nucleotide long conserved sequences. For each of them, we designed multiple A- and B-series toehold switches (Pardee et al, 2016), which are modified from original toehold switches to reduce translational leakage. Toehold switches are RNA sequences in the 5’-untranslated region of mRNA, which only allow the translation of a downstream protein coding sequence in the presence of their specific trigger. The B-series switches exhibit lower background signal, ideal for diagnostic kits, while A-series switches produce higher output signal, ideal for colorimetric assays. We wanted to test these different design principles to see which would better suit the needs of our kit.

Any toehold switches containing in-frame stop codons were disregarded from further consideration. We used a model adapted from Ma et al. (2018) to estimate the performance of the designed toehold switches (see our [Modeling page](https://2022.igem.wiki/aboa/model)). Three of the highest scoring sensors from each design series were taken into laboratory testing. We also collaborated with team TAU and their project TrigGate, who provided us alternative designs for each of our best toeholds. We ranked them with our model, and picked the highest scoring A-series and B-series toeholds for lab tests.

The toehold switch scores predicted by our model are lower than anticipated, as we did not yet have an optimized design algorithm when creating these. This issue was revised in later iterations of the design algorithm (see [Model](https://2022.igem.wiki/aboa/model) and [Partnership](https://2022.igem.wiki/aboa/partnership)). The theoretical maximum score is 54, yet this is almost impossible to reach in practise. In our experience based on evaluating viable toehold switches from literature, a score over 25 is to be pursued, but the scores rarely exceed 32. The modeling results for each toehold switch selected for laboratory testing is presented in Table 1.

**Table 1.** Results of computational modeling of toehold switches. We assigned a score for each toehold switch with our model, based on their deviations from ideal structures (see [Model](https://2022.igem.wiki/aboa/model)). Higher scores represent higher estimated performances.

| Toehold identifier | Score | Toehold identifier | Score |
| --- | --- | --- | --- |
| ABOA A70 | 16.41 | ABOA B39 | 13.19 |
| ABOA A92 | 15.40 | ABOA B46 | 14.08 |
| ABOA A95 | 15.39 | ABOA B69 | 11.99 |
| TrigGate A70 | 6.20 | TrigGate B46 | 24.07 |

## Successful Golden Gate assembly

---

Each sensor plasmid consists of the plasmid backbone, a promoter, a toehold switch, a gene encoding a reporter protein, and a terminator. pOdd1 was used as the plasmid backbone. We used the iGEM Type IIS standard fusion sites in our assembly, with slight modifications. The fusion site between the reporter coding sequence (cds) and the terminator was changed from GCTT to GGTT, as the standard junction site shared three consecutive nucleotides with the CGCT junction site between the terminator and the backbone. This way, the probability of incorrect assembly was lowered. Also, the toehold switch and reporter protein are joined with scarless assembly, but the junction site corresponds to the Type IIS standards. The promoter, the toehold switches, and the terminator were synthesized with flanking sequences producing these junction sites. The reporter genes were amplified from plasmids with PCR using primers with their corresponding flanking sites, so the amplified product also has these sites. An illustration of the complete sensor plasmid can be seen in Figure 1.

![A picture showing the structure of the toehold switch plasmids used in our project. The sites for promoter, toehold switch, reporter gene, terminator and antibiotic resistance can be seen from the picture. ](https://static.igem.wiki/teams/4207/wiki/results-figure1-plasmid-map.jpg)

**Figure 1.** Map of the sensor plasmids assembled with golden gate assembly. The kanamycin resistance was provided in the backbone, while other parts were added in the assembly. The modularity of our system is based on the exchangeable toehold switch in the sensor plasmid.

We assembled 14 sensor plasmids in total. A sensor plasmid with ꞵ-galactosidase as the reporter for each toehold and sensor plasmids containing our toehold switches and mScarlet-I as the reporter were assembled. In the assembly, an RFP gene is cleaved from the plasmid backbone and the remaining junction sites of the plasmid are not compatible so, in theory, a plasmid without inserts or the RFP gene cannot transform bacteria. This makes it possible to visualize the successfully transformed colonies as white, as they do not possess the RFP gene. To confirm the success of the assembly, we tested the presence of the insert in the white colonies with colony PCR. We used primers that attached to the linker sequence of the toehold sensor, as well as the terminator sequence. A representative result of the colony PCR reactions is shown in Figure 2.

![A picture of agarose gel electrophoresis for Golden Gate assembled sensor plasmids with m-Scarlet-I as the reporter. The gel contains six wells. All six sample wells show bright bands around 700-800 base pairs. No other bands are shown.](https://static.igem.wiki/teams/4207/wiki/results-figure2-results-geeli-b-ms.png)

**Figure 2.** Results of colony PCR reactions. Lane 1 was loaded with Quick-Load® Purple 1 kb Plus DNA Ladder (NEB, #N0550) and the molecular weight standards are marked. Lanes 2-7 contain colony PCR reactions performed for our novel toehold sensor plasmid with mScarlet-I as the reporter. The electrophoresis was performed in a 1 % agarose TAE gel with 100 V for 40 minutes.

In Figure 2 we can see that each sample in lanes 2-7 contains a PCR product with lengths between 700 and 800 bp. We expected PCR products of 762 bp. We can conclude that the assembly was successful, as we were able to amplify a sequence containing parts of three of the four inserts. All other constructs were confirmed in a similar fashion before being used in cell-free reactions.

## Validating the measurement technique

---

The activity of the produced β-galactosidase is assessed by measuring the absorbance at 420 nm, resulting from the breakdown of ONPG (2-nitrophenyl β-D-galactopyranoside) to 2-nitrophenyl and D-galactose. We planned to test the viability of our cell-free expression reactions by measuring the activity of β-galactosidase *in vivo*. We used an inducible pET-15b-derived expression plasmid containing the *lacZ* gene and induced its expression in BL21 (DE3) cell culture. ONPG was added to this cell culture and the activity of β-galactosidase was monitored by measuring absorbance at 420 nm. Absorbance was shown to increase over time, resulting from the breakdown of ONPG. This result validates our measurement technique for subsequent reactions.The results are presented in Figure 3.

![A graph of absorbance in 420 nm measures in cell-free reaction as a function of time (min) in BL21 (DE3). The absorbance values start from 0 at time point zero, and rise up to a little below 0,20. Error bars are also shown.](https://static.igem.wiki/teams/4207/wiki/results-figure3-invivo.png)

**Figure 3.** *In vivo* activity of β-galactosidase. The activity of β-galactosidase in BL21 (DE3) cell cultures was monitored by measuring absorbance at 420 nm, resulting from the breakdown of ONPG. The graph shows the increase of absorbance as the function of time. Each data point represents the average of four parallel reactions while error bars represent standard deviation.

## Demonstration of CFE viability

---

Cell-free expression (CFE) systems consist of all the necessary components needed for protein synthesis, but contain no live cells. There are three major components in a CFE system: protein components of transcription and translation, smaller functional molecules, such as NTPs and amino acids, and reaction buffers designed to maximize protein yield. To drive down the cost of making a CFE system, we opted to obtain the protein components from crude extracts. On top of this, we decided to use maltodextrin instead of using the traditional energy source 3-phosphoglyceric acid, as it has previously been proven to produce similar protein yields (Arce et al, 2021). In many applications of cell-free systems, the cells are lysed by bead-beating. The bead-beating tubes are single-use, which increases the cost of this method. Therefore, we tested two different cheaper methods for the cell lysis, sonication and autolysis. Sonication is a standard procedure in many protein extraction methods and the machine can be used multiple times, driving down the per-use cost (see [Design](https://2022.igem.wiki/aboa/design)).

In autolysis, the cells contain a plasmid that produces phage lambda endolysin. When exposed to freeze-thaw cycles, the inner membrane is disrupted and the endolysin gains access to the bacterial cell wall and degrades it, lysing the cell (Didovyk et al, 2017) (see [Design](https://2022.igem.wiki/aboa/design)).

We produced cell lysates by sonication (sCFE) and autolysis (aCFE) and compared their protein production capabilities in CFE systems. The functionality of the SCFE system was measured by its ability to produce β-galactosidase from the same expression plasmid as previously. The measurement setup was validated as before.

As we also wanted to test out mScarlet-I as the reporter in our toehold switches, we tested its expression from an expression plasmid in both lysates with otherwise identical CFE systems. Due to technical limitations, we measured the time required for each reaction to surpass the upper detection limit of our plate reader (10 000 A.U. over baseline). The sCFE systems required 41 ± 5,7 minutes, while the aCFE required 90 ± 48 minutes. The results from all of these measurements are presented in Figure 4.

![A. A graph of absorbance in 420 nm measures in cell-free reaction as a function of time (min) for autolysis (aCFE) and sonication (sCFE) samples. The absorbance values start from 0 at time point zero, aCFE rising to around 0,10, while sCFE rises to absorbance over 0,25 at time point 40, and then decreases to around 0,15 at time point 180. The error bars for both of the measurements are quite big, especially at time point 100 where they overlap with each other. B. A graph of fluorescence intensity in cell-free reaction as a function of time. Two samples for both autolysis (aCFE) and sonication (sCFE)c are shown. The sCFE samples reach 10 000 A.U. at around 40 min, while aCFE samples reach 10 000 A.U. at around 50 min and 120 min.](https://static.igem.wiki/teams/4207/wiki/results-figure4.png)

**Figure 4.** Comparison of different CFE systems. (A) The activity of β-galactosidase in CFE systems produced with autolysis (aCFE) or sonication (sCFE) was monitored by measuring the change of absorbance at 420 nm, resulting from the breakdown of ONPG. This graph shows the absorbance over time. Each data point represents the average of three independent reactions and error bars represent standard deviation. (B) Comparison of mScarlet-I production capabilities in CFE systems produced by autolysis and sonication. This graph shows the fluorescence intensity over baseline at 569/593 nm. The results are from two parallel measurements for both CFE systems.

In both measurements, sCFE exhibited more rapid signal production. These results suggest that sonication is a superior method for producing CFE systems, although repeating these tests is required to confirm this conclusion. However, absorbance in sCFE decreases after reaching a peak value, which could result from degradation of the colorimetric product, 2-nitrophenyl in the reaction. This would require further optimization of the colorimetric CFE reaction. Based on these measurements, we opted to use sCFE systems in all our subsequent reactions.

## Confirming toehold functionality in sCFE system

---

Next, we proceeded to test the sensors’ detection capabilities in the sCFE system. We used the 27B sensor from Pardee et al. (2016) (Addgene #75006) as our control to see if a proven toehold sensor could be activated in our system. The sensor produces β-galactosidase when activated, so we assessed its activity by measuring the absorbance at 420 nm as previously. We tested the activity in the presence of its specific ssDNA trigger. As we observed the toehold to be functional in our sCFE system, we proceeded to analyze the functionality of our novel toehold sensor plasmids, with β-galactosidase as the reporter, and compare their performance to the control teohold sensor. The results are shown in Figure 5.

![A. A graph of absorbance in 420 nm measures in cell-free reaction as a function of time (min) in sample 27B with and without trigger. The absorbance values start from 0 at time point zero, sample 27B with trigger rising up to around 0.12 and sample 27B without trigger rising up to around 0,05 at time point 180. Differences between the samples start to accumulate around time point 30. The error bars for 27B with trigger are big, especially in time point 180 where the error bars go from 0.05 up to 0.20. B. A graph of absorbance in 420 nm measures in cell-free reaction as a function of time (min) in sample A70 with and without trigger. The absorbance values start from 0 at time point zero, sample A70 without trigger rising to around 0.04, sample A70 without trigger rising up to around 0.08 at time point 180. The samples start to deviate from each other at around 30 minutes. Both of the samples have some error margin, the error bars of sample A70 with trigger almost reaching the error bars of A70 without sample. C. A graph of absorbance in 420 nm measures in cell-free reaction as a function of time (min) in sample B46 with and without trigger. The absorbance values start from 0 at time point zero, sample B46 without trigger rising up to around 0.07 while B46 with trigger rises up to around 0.04 at time point 180. The samples start to deviate from each other at around time point 100. The error bars for B46 without trigger are notably large, B46 with trigger also shows some error. D. A graph of absorbance in 420 nm measures in cell-free reaction as a function of time (min) in sample TG70 with and without trigger. The absorbance values start from 0 at time point zero, TG70 without trigger rising up to around 0.11 and TG70 rising up to 0.02. The samples deviate from each other especially at time point 110, where the absorbance values of TG70 with trigger drop drastically. The error bars for TG70 with trigger are large. E. A graph of absorbance in 420 nm measures in cell-free reaction as a function of time (min) in sample TG46 with and without trigger. The absorbance values start from 0 at time point zero, TG46 without trigger rising up to around 0.12 and TG46 with trigger rising up to around 0.18. No error bars are shown for either of the samples. F. A bar chart showing ON/OFF-ratios of A70, B46, TG70, TG46 and 27B. A70 and B46 show a ratio around 0.5. TG70 shows a ratio of below 0.25. TG46 shows a ratio of nearly 1.5, while 27B shows a ratio of nearly 2.5.](https://static.igem.wiki/teams/4207/wiki/results-figure5.png)

**Figure 5.** Toehold switch performance in sCFE system. sCFE reactions containing toehold sensor plasmids with β-galactosidase as the reporter protein were monitored by measuring the absorbance at 420 nm. Cell-free reactions containing (A) the control toehold sensor plasmid and (B-E) our novel toehold sensor plasmids were measured in the presence or absence of their specific trigger ssDNA. Graphs A-E show the absorbance over time in the reactions. Each data point represents the average of two parallel reactions and error bars represent standard deviation. Graph E shows only the average of two independent reactions as representative results. Graph F shows the ON/OFF ratios for each toehold switch based on these reactions, calculated as the ratio of end point signal of reactions with and without trigger. The average of the two parallel reactions were used in the calculation.

The established 27B toehold sensor plasmid showed clear distinction in absorbance produced in the presence and absence of its specific ssDNA trigger. The reactions containing the trigger produced a 2.46-fold end point signal compared to the reactions where the trigger was left out (ON/OFF-ratio). This result validates the sCFE system for being capable of toehold switch-based sensing. However, the ON/OFF-ratio measured in these tests was lower than in published CFE systems (Arce et al, 2021; Pardee et al, 2016), which suggests that our sCFE system is not yet optimized for this purpose.

Our novel toehold sensor plasmids did not match the performance of the control toehold switch in our measurements. Toehold switches A70, B46 and TG70 were not observed to exhibit trigger-dependent signal production, as their signal production was lower in the presence of the trigger; with ON/OFF-ratios of 0.56, 0.56 and 0.17, respectively. Our toehold sensor B46 was seen to exhibit trigger-specific signal production, as it had an ON/OFF-ratio of 1.45 in our tests. These results are still limited and further tests would be needed to confirm functionality. On top of this, the remaining eight of the assembled toehold sensor plasmids would require testing to draw further conclusions of the functionality of our system.

As we gained experimental data about the functionalities of the toehold switches, we assessed the performance prediction capability of our model. We plotted the ON/OFF-ratios of our toehold switches as the function of their scores assigned by our model. A clear correlation between the predicted and observed performance can be seen, which suggests our model to be viable in predicting the best performing toehold switches in identical conditions. However, we do not have enough data to credibly draw any conclusions. The results are presented in Figure 6.

![A graph showing the ON/OFF-ratio as a function of score. Four points are presented in the graph, and a linear fit line is drawn between the points. The second point is exactly on the line, while other points deviate a bit.](https://static.igem.wiki/teams/4207/wiki/results-figure6-model-perfomance.png)

**Figure 6.** Correlation of predicted and measured performance of toehold switches. The graph shows the ON/OFF-ratio, measured from two independent reactions with and without the trigger, over the score predicted by our model.

As we had demonstrated the viability of our sCFE system for toehold-based sensing using ssDNA as the trigger, we wanted to test the system for RNA sensing. Our goal was to enable sensing of NASBA amplicons, which would be produced from the sample to be analyzed (see [Proposed Implementation](https://2022.igem.wiki/aboa/implementation)). To test this, we sought to analyze the control toehold sensor’s ability to sense RNA sequence designed to mimic NASBA amplicons. However, the control toehold switch was not observed to exhibit RNA trigger-dependent signal production. The result of this measurement is shown in Figure 7.

![A graph of absorbance in 420 nm measures in cell-free reaction as a function of time (min) in sample 27B with and without sample. The absorbance values start from 0 at time point zero, and go up to approximately 0.15 in both samples. The sample 27B with trigger, seems to have a bit higher absorbance especially from 100 minutes to 180 minutes, where the measurement stops. The error bars for both samples at this time are very high.](https://static.igem.wiki/teams/4207/wiki/results-figure7-zs-rna.png)

**Figure 7.** Assessment of RNA-sensing in sCFE. The production of β-galactosidase is measured by the increase of absorbance at 420 nm, resulting from breakdown of ONPG. The absorbance is measured from reactions in the presence and absence of RNA trigger. Absorbance is presented as the function of time, with each data point representing the average of two independent reactions and error bars representing standard deviation.

These results suggest that our sCFE system inhibits the sensing of RNA sequences. However, additional testing would be required, as no concrete conclusions can be drawn from the result. Here we have only measured two reactions, which exhibited high variability, seen as the high values of standard deviations.

As the functionality of our toehold-switches in the presence of RNA triggers was not demonstrated, we moved to investigating their function in a commercial PURE (Protein Expression using Recombinant Elements) system (Shimizu et al, 2001). The reconstituted nature of PURE systems leads to fewer contaminants in the reactions. Testing the control sensor construct as well as our novel constructs in the PURE system would give us insights on optimizing our sCFE system. The 27B control construct was validated using a commercial PURExpress system (NEB #E6800) (Pardee et al, 2016) so we sought to replicate this result. However, we were not able to measure any transcription-translation activity in our measurements. Thus, we did not gain any additional data about our sensor constructs or the sCFE system with these measurements.

## Discussion

---

We succeeded in producing a viable CFE system capable of toehold-based sensing. We managed to create functional toehold sensors and demonstrated their effectiveness in trigger-dependent translation. However, our goal of RNA trigger sensing was not showcased and based on our test reactions, the performance of our toehold sensors was not on par with the control sensor. The data set is limited and more information would need to be gathered to speculate the system’s functionality in more detail. Still, we gained valuable information and were able to pinpoint deficiencies in our system, which could be revised in ensuing revisions.

Our toehold switches were not demonstrated to exhibit high trigger-dependency in translational activity. This could stem from insufficient performance of the designed toehold switches. Our design algorithm was not optimized when creating these toehold switches and in later iterations, we were able to produce better performing toehold switches, according to our model. We also gained some experimental data about our model’s viability. With further testing of the assembled toehold sensors and testing of new toehold switches produced by updated versions of our algorithm and team TAU’s TrigGate, we would further optimize the toehold-mediated sensing. On top of the toehold switch, other parts of the sensor plasmid would need optimization as well.

The control toehold switch also displayed lower translational specificity for its trigger, which suggests that our sCFE inhibits the sensing capabilities of toehold switches. Again, further testing with this toehold sensor as well as other established sensors would be necessary for examining this suggestion. Our sCFE system was produced with crude extract from sonication, while established toehold sensing in CFE systems have been conducted in systems produced with crude extract from bead-beating (Arce et al, 2021) or with PURE (protein expression using recombinant elements) systems (Green et al, 2017; Pardee et al, 2016). On top of this, the extract-based CFE was produced with a strain deficient of multiple nucleases. As a result, the established systems inherently contain lower amounts of nucleases, which can impair the stability of the sensor DNA and RNA. The sensor DNA was linearized before input in these systems, which could also impact the transcriptional activity and therefore the amount of sensor RNA in the reaction.

Sensing of RNA triggers was not observed in our sCFE system. This was also documented in published CFE systems. This could be caused by faulty produced RNA-triggers as we were unable to confirm correct transcription products due to unavailability of necessary components of RNA analysis. Our system could also impair the stability of the RNA trigger, which would hinder signal production. However, we did not perform enough experiments for us to draw credible conclusions.

We also observed a decrease in the colorimetric signal in sCFE reactions. This was not the case in previously established CFE systems used for toehold-mediated sensing (Pardee et al, 2016; Ma et al, 2018; Arce et al, 2021). As we’d visualize our system to produce a colorimetric signal, degradation of the colorimetric product impairs the viability of the system. This could be solved by optimizing the CFE system to increase the stability of 2-nitrophenyl. This issue could also be solved by changing the substrate, for example to chlorophenol red-β-D-galactopyranoside (CPRG), which was used in previous systems. This could also produce a signal more easily detected visually, as the color change is more dramatic with CPRG (yellow to dark red) than in ONPG (colorless to yellow).

Despite these challenges, our detection system shows promise for its capability. Overcoming the issues and optimization of all parts of the system would provide a powerful detection system, which could help fight the global issues of hunger and food security.

## References

---

* Arce, A., Guzman Chavez, F., Gandini, C., Puig, J., Matute, T., Haseloff, J., Dalchau, N., Molloy, J., Pardee, K., & Federici, F. (2021). Decentralizing Cell-Free RNA Sensing With the Use of Low-Cost Cell Extracts. *Frontiers in Bioengineering and Biotechnology*, 9, 744. https://doi.org/10.3389/FBIOE.2021.727584/BIBTEX

* Didovyk, A., Tonooka, T., Tsimring, L., & Hasty, J. (2017, August 21). Rapid and Scalable Preparation of Bacterial Lysates for Cell-Free Gene Expression. *ACS Synthetic Biology*, 6(12), 2198–2208. https://doi.org/10.1021/acssynbio.7b00253

* Green, A. A., Kim, J., Ma, D., Silver, P. A., Collins, J. J., & Yin, P. (2017). Complex cellular logic computation using ribocomputing devices. *Nature* 2017 548:7665, 548(7665), 117–121. https://doi.org/10.1038/nature23271

* Green, A. A., Silver, P. A., Collins, J. J., & Yin, P. (2014). Toehold Switches: De-Novo-Designed Regulators of Gene Expression. *Cell*, 159(4), 925–939. https://doi.org/10.1016/J.CELL.2014.10.002

* Ma, D., Shen, L., Wu, K., Diehnelt, C. W., & Green, A. A. (2018). Low-cost detection of norovirus using paper-based cell-free systems and synbody-based viral enrichment. *Synthetic Biology*, 3(1). https://doi.org/10.1093/SYNBIO/YSY018

* Pardee, K., Green, A. A., Takahashi, M. K., Braff, D., Lambert, G., Lee, J. W., Ferrante, T., Ma, D., Donghia, N., Fan, M., Daringer, N. M., Bosch, I., Dudley, D. M., O’Connor, D. H., Gehrke, L., & Collins, J. J. (2016). Rapid, Low-Cost Detection of Zika Virus Using Programmable Biomolecular Components. *Cell*, 165(5), 1255–1266. https://doi.org/10.1016/J.CELL.2016.04.059

* Shimizu, Y., Inoue, A., Tomari, Y., Suzuki, T., Yokogawa, T., Nishikawa, K., & Ueda, T. (2001). Cell-free translation reconstituted with purified components. Nature biotechnology, 19(8), 751–755. https://doi.org/10.1038/90802

{% endblock %}