iGEM 2022 — Team tu-braunschweig

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Notebook

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## July

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13.07.2022 - 14.07.2022:

* first start in the lab
* preparation of first media, buffers and reagents

15.07.2022:

* our chemo competitor cells have arrived. We started with a transformation for testing
* creating glycerol stocks from DH5α
* eYFP and mTurq chemical transformation in DH5α

18.07.2022 - 19.07.2022:

* precultured our transformation colonys
* plasmid preapration with 6 colonies from our transformation

19.07. - 20.07.2022:

* PCR amplification of the amplification component pNpuC\_CTEV\_caged\_insert
* 1% gel electrophorese

25.07.2022 - 26.07.2022:

* PCR amplification of the amplification component pNpuN\_NTEV\_insert
* 1% gel electrophorese
* PCR Clean up

27.07.2022:

* PSB1C3 chemical transformation in DH5α strain (psB1C3 BBa\_I20270 & psB1C3 BBa\_J364000)

28.07.2022 - 29.07.2022:

* precultivation from PSB1C3 transformation colonys
* plasmid preparation

## August

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02.08.2022:

* restriction digest for the amplification component
* digestion of the insert using NpuN and NpuC with restirction enzymes SpeI and PstI
* digestion of the vector PSB1C3 for amplification component
* incubation for 1.5 hours

05.08.2022:

* digestion of the vector PSB1C3 for amplification component
* PCR from eYFP and mTurq

10.08.2022 - 11.08.2022:

* PCR product from eYFP and mTurq (template: PSB1C3) was digested with Dpn1 according to attached protocol
* PCR product from eYFP and mTurq was eluted from the gel

12.08.2022:

* starting gibson assembly with eYFP and mTurq in the vector PSB1C3

23.08.2022 - 25.08.2022:

* after the Gibson assembly a colony PCR from different colonys was performed
* after the colony PCR the product was eluted in a 1% agarose gel

26.08.2022:

* three colonys where picked for protein production (colony 2, 6 and 8)
* precultured colonys 2, 6 and 8 with following plasmid preparation

27.08.2022:

* plasmid preparation from colony 2, 6 and 8
* preparing samples for sequencing the parts

29.08.2022 - 30.08.2022:

* precultured colonys for protein production over night
* first protein production of FRET pair
* testing the fluoreszence via special filters
* second restriction digest for amplification component with differnet enzymes producer

## September

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13.09.2022:

* PCR of NpuN and NpuC first PCR was made to add the his-tag at the ORF (long)
* PCR product was eluted in 1% agarose gel

14.09.2022:

* preparing new primers for gibson assembly
* PCR from R\_his\_Ntev, R\_his\_Ctev, R\_his\_Ftev, f\_p\_TEV\_complete\_insert, F\_his\_NFtev, F\_his\_Ctev

15.09.2022 - 17.09.2022:

* adjust FRET protein production by different factors, such as shaking speed
* for gibson assembly the backbone needs to be first opened
* therefore already FRET-Plasmid (Reporter Component was) purified. It will now be opened by a PCR with two Primers

17.09.2022 - 18.09.2022:

* transformation of the performed assembly
* after the transformation a pre culture was started

19.09.2022:

* plasmid preparation of the intein part (amplification component)
* after the plasmid prep a colony PCR with an gel electrophoreses were done

22.09.2022 - 23.09.2022:

* PCR of FTEV short and FTEV long
* precultured colony from FTEV short and FTEV long
* plasmid preparation and preparation for sequencing

## Future plans

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* purification of proteins with HIS-tag
* measurement of the FRET signal on the tecan reader
* testing of the components purified and in cell lysate
* quantification of the test with time progression of the FRET signal

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Engineering

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#

"The way to succeed is to double your failure rate"
- Thomas J. Watson, pioneer in the development computing equipment for IBM

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The design process is iterative, which means that steps are repetet as many times as necessary. Making improvements along the way and to learn from failure and uncover new design possibilities.

## Disclaimer: Engineering cycle2

On this site the engineering process is described only for our hardware. To see the engineering process for our model please have a look at our [model site](https://2022.igem.wiki/tu-braunschweig/model).

## First run of the engineering cycle

The purpose of the box was to create a dark space for fluorescence detection into which a microtiter plate containing the sample/the samples and the lysates of our detection system can be inserted by means of a drawer. Through an opening on the top of the box, photos can be taken with a cell phone with a flash light function. Due to the filters in front of the flash as well as the camera, certain wavelengths can be detected. The cell phone attachment is interchangeable and thus the box can be used with different cell phones. After the initial design of the box was created, the first prototype was 3D printed. When the first version was tested, we encountered several problems: First, because of the camera position on the cell phone, the drawer had to be redesigned to get a photo that shows the necessary area. Secondly, we have established that the drawer is too small for the microtiter plate we have planned to use.

![](https://static.igem.wiki/teams/4184/wiki/6.png)

## Second run of the engineering cycle

In the second version, attention was paid to the usage of less material as well as fixing all the flaws we identified in the first engineering cycle. This allows resource-saving production of the box, as well as a less expensive and lightweight design. The second version was printed and then tested by a physician. He recommends a reduction in the steps required to use the box to facilitate the application in a medical practice and make it easier to understand for everyone.

![](https://static.igem.wiki/teams/4184/wiki/7.png)

![](https://static.igem.wiki/teams/4184/wiki/hardware/bilderbox/box.jpg)

An insight into the successful application is provided here: [proof-of-concept](https://2022.igem.wiki/tu-braunschweig/proof-of-concept)

## Third run of the engineering cycle

Through the feedback from a medical professional, the second version of the box was redesigned. To implement the tips of the professional, it had to be modified so that fewer steps are needed to detect antigens. Therefore, the third version of the box takes over the individual pipetting steps by pumping the lysates into a Petri dish inside the box, when pushing a button. This makes the use of the box more understandable for everyone and enables a quick operation by physicians.

Therefore, the third version of the box was designed to allow access to the inside of the box. Since we are dealing with three different lysates and therefore three different pumps with tubes, an adapter was printed that connects the three tubes to one passage that leads to the Petri dish inside the box. The electrical system is stored separately outside the box to maintain physical separation between the sensitive electrical elements and the microtiterplate. A circuit diagram was created and the pumps were programmed. The final version of the box was then 3D printed and the pumps installed. After 3D printing, the box got successfully tested.

A video of the pump function can be seen here: [Hardware](https://2022.igem.wiki/tu-braunschweig/hardware)

![](https://static.igem.wiki/teams/4184/wiki/ec-3.png)

![](https://static.igem.wiki/teams/4184/wiki/hardware/whatsapp-image-2022-10-12-at-00-44-44-4.jpeg)

![](https://static.igem.wiki/teams/4184/wiki/whatsapp-image-2022-10-12-at-17-04-32.jpeg)

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Experiments

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### Agarose Gel Electrophoresis

**Materials**

* Agarose* SERVA DNA Stain Clear G* 1x TAE-buffer* 40mM TRIS (Tris-(hydroxymethyl)-aminomethane)* 20mM Acetat* 1mM EDTA (Ethylenediaminetetraacetic acid)

**Procedure**

* mix 1g Agarose with 100 ml 1x TAE in a flask* microwave the mixture until Agarose is completely dissolved (mixture is clear), the cap should sit loose on top of the flask to prevent the loss of water by vaporization and pressure-build up* add 5 ul (to 100 ml solution) of SERVA DNA Stain Clear G and swirl the flask a few times by hand / add 2 drops of EtBr per 50 ml solution* pour the liquid agarose gel into the gel tray and wait for the gel to solidify* insert gel tray into gel box after removing gel casting stand or tape* remove comb* load the DNA samples into the gel pockets using a pipette* run the gel by connecting the voltage generator to the gel box* set the voltage generator to 85 - 140 V

form: Thermo Fischer Scientific

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### Chemical Transformation(*E. coli*)

**Materials**

* Selection plates (LBsolid + antibiotic)* SOC/ LB liquid* Chemo competent *E. coli** Plasmid DNA (1 pg - 100 ng)* Icebox* Preheated Thermobloc/ Water bath (42 °C)* Preheated Thermoshaker (37 °C)* Table Centrifuge for Reaction tubes (1.5 mL)* Sterile Drigalski spatula/ Plating beads* Pipettes and Pipet tips (1000 µL, 200 µL)

**Procedure**

* Thaw 1 Aliquot (50 µL) of chemo-competent *E. coli* cells on ice for 10 minutes. Thaw on ice until the last ice crystals disappear* Add 1 pg - 100 ng of plasmid DNA (1 - 5 µL) to cells and mix without vortexing* Place on ice for 30 minutes* Heat shock at 42 °C for 45 - 60 seconds* Place on ice for 5 minutes* Add 950 µL of room temperature liquid SOC or LB* Place at 37 °C for 60 minutes. Shake vigorously (250 rpm)* Spread 200 µL on selection plates (LB solid + AB concentration) and plate with the help of a drigalski spatula/ plating beads* Centrifuge until there is a cell pellet (ca. 1-2 min.) and discard the supernatant up to 50 µL* Resuspend the cell pellet in the 50 µL and plate with the help of a drigalski spatula/ plating beads* Incubate the selection plates at 37 °C for ca. 12 hours (over night)* Count the transformed *E. coli* colonies and calculate the transformation efficiency with the formula (TE) = Colonies/µg/Dilution

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### PCR using Q5 polymerase

**Materials**

* Thermocycler* PCR tubes* Pipette* Q5 Polymerase* primer* DNA Template* 5x Q5 reaction buffer* dNTPs* bidest. H2O

**Procedure**

Mastermix:

|  |  |  |
| --- | --- | --- |
| Component | 25 ul | Final Concentration |
| Q5 High-Fidelity 2X Master Mix | 12,5 ul | 1X |
| 10 uM Forward Primer | 1,25 ul | 0,5 uM |
| 10 uM Reverse Primer | 1,25 ul | 0,5 uM |
| Template DNA |  | 1 pg - 1 ng |
| Nuclease Free Water | to 25 ul |  |

Thermocycler Conditions:

|  |  |  |  |
| --- | --- | --- | --- |
| Step | Temperature | Time |  |
| Initial Denaturation | 98°C | 30 sec. |  |
| Denaturation | 98°C | 5-10 sec. | 30-35 cycles |
| Annealing | 60°C | 10-30 sec. | 30-35 cycles |
| Elongation | 72°C | 30 sec./ 1kb | 30-35 cycles |
| Final Elongation | 72°C | 2 minutes |  |
| Hold | 4°C |  |  |

from: New England Biolabs GmbH

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### Plasmid Isolation Alkaline Lysis (*E. coli*)

**Materials**

* Buffer 1:* 50 mM Tris-HCl, pH 8.0; 10 mM EDTA; 100 mg/mL RNase A, (store at 4˚C)* Buffer 2:* 0.2 M NaOH; 1 % (w/v) SDS* Buffer 3:* 3 M Potassium acetate, pH 5.5

**Procedure**

* Picking of an *E.coli* colony and inoculation of 3-5 mL LB liquid + appropriate antibiotic into a test tube.* incubation at 37 °C and 150 - 200 rpm overnight* The next day, transfer 1.5 mL of each preculture into 2.0 mL of eppis* Centrifuge for 1 -2 minutes at 13,000 rpm* Discard supernatant and centrifuge again for 30 seconds at 13,000 rpm* Thoroughly pipette off the entire supernatant* Pipette 100 µL of solution 1 onto the pellet and resuspend by vortexing (Thorough).* Incubate the eppis at room temperature for 5-7 min.* Add 200 µL solution 2 and CAREFULLY invert (Do not vortex!) -> solution should clear up* Incubate on ice for 2-3 minutes* Add 150 µL solution 3 (precipitation of DNA and proteins) and invert* Centrifuge for 15 minutes at 16,000 rpm (optimal 14 °C, RT also ok)* Pipette supernatant carefully into a 1.5 mL eppi (do not take a pellet)* First wash step with 100 % ethanol 1000 µL* Invert, centrifuge at 16,000 rpm for 12 min.* Discard supernatant and perform 2nd wash step with 70 % ethanol at 16,000 rpm for 5 minutes* Remove supernatant (pipette off) and dry pellets* Dissolve the pellets in 30 µL nuclease-free water (+RNase).* Measure the DNA concentration on the NanoDrop (note c in ng/ µL on the eppi page).* Freeze dissolved plasmid DNA

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### NEBuilder Hifi NDA Assembly

|  |  |
| --- | --- |
|  | 2-3 Fragment Assembly |
| Recommended DNA Molar Ration | vector:insert = 1:2 |
| Total amount of fragments | 0.03-0,2 moles X ul |
| NEBuilder Hifi DNA Assembly Master Mix | 10 ul |
| Deionized H2O | 10-X ul |
| Total Volume | 20 ul |

* Incubate samples at 50°C for 60 minutes

from: New England Biolabs GmbH

**Protein Expression in strain KRX**

* Grow starter cultures overnight in LB medium (with antibiotic) at 37°C and 275 rpm* Dilute overnight starter cultures 1:100 into LB containing antibiotic* Grow cultures at 37°C and 275 rpm until (O.D.600) of 0.4–0.5* Shift cultures to another incubator shaker set at 15–25°C, and continue shaking at approximately 275 rpm.* When the cultures reach an O.D.600 of 0.5–0.6 for LB, induce protein expression by adding rhamnose to a concentration of 0.1% (1:200 dilution of 20% rhamnose)* Grow cultures overnight at 15–25°C and 275 rpm* Harvest cells by centrifugation (e.g., 10,000 × *g* for 3 minutes in a microcentrifuge or 4,000 × *g* for 10 minutes in a larger format

from: Promega

### Recipes for media/buffers/solutions

**LB solid:**

10 g peptone/triptone

5 g yeast extract

10 g sodium chloride

ad 1 L and autoclave

**Solution 1** (alkaline lysis):

9 g glucose

25 mL 1M Tris-HCl (pH 8.0)

20 mL 0.5M EDTA (pH 8.0)

ad 1 L sterile dH2O

not autoclave!

**Solution 2** (Alkaline Lysis):

100 mL SDS stock solution (1 %)

8 g sodium hydroxide

ad 1 L sterile dH2O

not autoclave!

**Solution 3** (Alkaline lysis):

600 mL 5M potassium acetate

115 mL glacial acetic acid

ad 1 L sterile dH2O!

not autoclave!

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Results

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# Text Box

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## Results

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We succesfully created and cloned three plasmids (see parts list to see how they are composed). Final evaluation of the plasmids will be done by sequencing.

Additionally we created a hardware component ( as you can see in the hardware section) and have a great model (please have a look at our model site). The individual results are shown and discussed in their corresponding sites.

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