iGEM 2018 — Team Valencia_UPV

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# Team:Valencia UPV/Notebook

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[HOME](https://2018.igem.org/Team%3AValencia_UPV)

TEAM

[Team Members](https://2018.igem.org/Team%3AValencia_UPV/Team)
[Collaborations](https://2018.igem.org/Team%3AValencia_UPV/Collaborations)

PROJECT

[Description](https://2018.igem.org/Team%3AValencia_UPV/Description)
[Design](https://2018.igem.org/Team%3AValencia_UPV/Design)
[Experiments](https://2018.igem.org/Team%3AValencia_UPV/Experiments)
[Notebook](https://2018.igem.org/Team%3AValencia_UPV/Notebook)
[InterLab](https://2018.igem.org/Team%3AValencia_UPV/InterLab)
[Model](https://2018.igem.org/Team%3AValencia_UPV/Model)
[Results](https://2018.igem.org/Team%3AValencia_UPV/Results)
[Demonstrate](https://2018.igem.org/Team%3AValencia_UPV/Demonstrate)
[Improve](https://2018.igem.org/Team%3AValencia_UPV/Improve)
[Attributions](https://2018.igem.org/Team%3AValencia_UPV/Attributions)

PARTS

[Parts Overview](https://2018.igem.org/Team%3AValencia_UPV/Parts)
[Basic Parts](https://2018.igem.org/Team%3AValencia_UPV/Basic_Part)
[Composite Parts](https://2018.igem.org/Team%3AValencia_UPV/Composite_Part)
[Part Collection](https://2018.igem.org/Team%3AValencia_UPV/Part_Collection)

[SAFETY](https://2018.igem.org/Team%3AValencia_UPV/Safety)

HUMAN PRACTICES

[Human Practices](https://2018.igem.org/Team%3AValencia_UPV/Human_Practices)
[Education & Engagement](https://2018.igem.org/Team%3AValencia_UPV/Public_Engagement)

AWARDS

[Applied Design](https://2018.igem.org/Team%3AValencia_UPV/Applied_Design)
[Entrepreneurship](https://2018.igem.org/Team%3AValencia_UPV/Entrepreneurship)
[Hardware](https://2018.igem.org/Team%3AValencia_UPV/Hardware)
[Measurement](https://2018.igem.org/Team%3AValencia_UPV/Measurement)
[Model](https://2018.igem.org/Team%3AValencia_UPV/Model)
[Plant](https://2018.igem.org/Team%3AValencia_UPV/Plant)
[Software](https://2018.igem.org/Team%3AValencia_UPV/Software)

[JUDGING FORM ⇗](https://igem.org/2018_Judging_Form?team=Valencia_UPV)

# Notebook

Document the dates you worked on your project. This should be a detailed account of the work done each day for your project.

### What should this page have?

* Chronological notes of what your team is doing.
* Brief descriptions of daily important events.
* Pictures of your progress.
* Mention who participated in what task.

### Inspiration

You can see what others teams have done to organize their notes:

* [2014 ATOMS-Turkiye](https://2014.igem.org/Team%3AATOMS-Turkiye/Notebook)
* [2014 Tec Monterrey](https://2014.igem.org/Team%3ATec-Monterrey/ITESM14_project.html#tab_notebook)
* [2014 Kyoto](https://2014.igem.org/Team%3AKyoto/Notebook/Magnetosome_Formation#title)
* [2014 Cornell](https://2014.igem.org/Team%3ACornell/notebook)


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

# Team:Valencia UPV/Experiments

[![](https://static.igem.org/mediawiki/2018/8/8a/T--Valencia_UPV--pruebaPrinteriaWhiteUPV2018.png)](https://2018.igem.org/Team%3AValencia_UPV)

* Team

  + [Team](https://2018.igem.org/Team%3AValencia_UPV/Team)
  + [Attributions](https://2018.igem.org/Team%3AValencia_UPV/Attributions)
  + [Collaborations](https://2018.igem.org/Team%3AValencia_UPV/Collaborations)
  + [Gallery](https://2018.igem.org/Team%3AValencia_UPV/Gallery)
* Project

  + [Description](https://2018.igem.org/Team%3AValencia_UPV/Description)
  + [Design](https://2018.igem.org/Team%3AValencia_UPV/Design)
  + [Experiments](https://2018.igem.org/Team%3AValencia_UPV/Experiments)
  + [Inter Lab](https://2018.igem.org/Team%3AValencia_UPV/InterLab)
  + [Safety](https://2018.igem.org/Team%3AValencia_UPV/Safety)
  + [Improve](https://2018.igem.org/Team%3AValencia_UPV/Improve)
  + [Demonstrate](https://2018.igem.org/Team%3AValencia_UPV/Demonstrate)
  + [Modeling](https://2018.igem.org/Team%3AValencia_UPV/Model)
  + [Hardware](https://2018.igem.org/Team%3AValencia_UPV/Hardware)
  + [Software](https://2018.igem.org/Team%3AValencia_UPV/Software)
  + [Graphic Design](https://2018.igem.org/Team%3AValencia_UPV/GraphicDesign)
* Parts

  + [Parts](https://2018.igem.org/Team%3AValencia_UPV/Parts)
  + [Part Collection](https://2018.igem.org/Team%3AValencia_UPV/Part_Collection)
* Results

  + [Results](https://2018.igem.org/Team%3AValencia_UPV/Results)
  + [Demonstrate](https://2018.igem.org/Team%3AValencia_UPV/Demonstrate)
  + [Improve](https://2018.igem.org/Team%3AValencia_UPV/Improve)
* Human

  + [Human Practices](https://2018.igem.org/Team%3AValencia_UPV/Human_Practices)
  + [Integrated Human](https://2018.igem.org/Team%3AValencia_UPV/Human_Practices#Integrated)
  + [Public Engagement](https://2018.igem.org/Team%3AValencia_UPV/Public_Engagement)
* [Medals](https://2018.igem.org/Team%3AValencia_UPV/Medals)

[![](https://static.igem.org/mediawiki/2018/8/8a/T--Valencia_UPV--pruebaPrinteriaWhiteUPV2018.png)](https://2018.igem.org/Team%3AValencia_UPV)

[![](https://static.igem.org/mediawiki/2018/b/b9/T--Valencia_UPV--iconHardwareUPV2018.png)](https://2018.igem.org/Team%3AValencia_UPV/Hardware)
[![](https://static.igem.org/mediawiki/2018/d/d1/T--Valencia_UPV--iconSoftwareUPV2018.png)](https://2018.igem.org/Team%3AValencia_UPV/Software)[![](https://static.igem.org/mediawiki/2018/f/f6/T--Valencia_UPV--iconDesignUPV2018.png)](https://2018.igem.org/Team%3AValencia_UPV/Design)[![](https://static.igem.org/mediawiki/2018/0/0e/T--Valencia_UPV--iconModellingUPV2018.png)](https://2018.igem.org/Team%3AValencia_UPV/Model)

* Team

  + [Team](https://2018.igem.org/Team%3AValencia_UPV/Team)
  + [Attributions](https://2018.igem.org/Team%3AValencia_UPV/Attributions)
  + [Collaborations](https://2018.igem.org/Team%3AValencia_UPV/Collaborations)
  + [Gallery](https://2018.igem.org/Team%3AValencia_UPV/Gallery)
* Project

  + [Description](https://2018.igem.org/Team%3AValencia_UPV/Description)
  + [Design](https://2018.igem.org/Team%3AValencia_UPV/Design)
  + [Experiments](https://2018.igem.org/Team%3AValencia_UPV/Experiments)
  + [Model](https://2018.igem.org/Team%3AValencia_UPV/Model)
  + [Hardware](https://2018.igem.org/Team%3AValencia_UPV/Hardware)
  + [Software](https://2018.igem.org/Team%3AValencia_UPV/Software)
  + [Inter Lab](https://2018.igem.org/Team%3AValencia_UPV/InterLab)
  + [Safety](https://2018.igem.org/Team%3AValencia_UPV/Safety)
  + [Graphic Design](https://2018.igem.org/Team%3AValencia_UPV/GraphicDesign)
* Results

  + [Results](https://2018.igem.org/Team%3AValencia_UPV/Results)
  + [Improve](https://2018.igem.org/Team%3AValencia_UPV/Improve)
  + [Demonstrate](https://2018.igem.org/Team%3AValencia_UPV/Demonstrate)
* Parts

  + [Parts](https://2018.igem.org/Team%3AValencia_UPV/Parts)
  + [Part Collection](https://2018.igem.org/Team%3AValencia_UPV/Part_Collection)
* Human

  + [Human Practices](https://2018.igem.org/Team%3AValencia_UPV/Human_Practices)
  + [Integrated Human](https://2018.igem.org/Team%3AValencia_UPV/Human_Practices#Integrated)
  + [Public Engagement](https://2018.igem.org/Team%3AValencia_UPV/Public_Engagement)
* Awards

  + [Medals](https://2018.igem.org/Team%3AValencia_UPV/Medals)
  + [Awards](https://2018.igem.org/Team%3AValencia_UPV/Awards)
* [PRESS & CONTACT](https://2018.igem.org/Team%3AValencia_UPV/PressAndContact)

Stack Multipurpose HTML Template

![](https://static.igem.org/mediawiki/2018/thumb/d/da/T--Valencia_UPV--ExperimentsTextoUPV2018.jpeg/1200px-T--Valencia_UPV--ExperimentsTextoUPV2018.jpeg.png)

#### Index

* [Printeria protocols & experiments](#imPrinteria)
* [Parts characterization](#imCharact)
* [Reporter proteins comparisons](#imReporter)

During the development of Printeria, experimentation has been one of the fundamental aspects in each step of our project. The design of new experiments and protocols has allowed us to validate the results obtained from the device or the laboratory with the theoretical schemes and to guarantee the repeatability of each experience. These experiments were intended to cover three key objectives of the project:

* To demonstrate the functioning of the Printeria device through the **concept tests** of the technologies separately and through the **tests and protocols** related to Printeria established in the Wet Lab.
* **To characterize the parts** in an experimental way in order to include the maximum information in our Printeria [Part Collection](https://2018.igem.org/Team%3AValencia_UPV/Part_Collection).
* To obtain as much information as possible about the **reporter proteins** commonly used in Web Lab experiments, such as GFP, YFP or sfGFP.

**Check the results of our experiments yourself...**

### Printeria protocols & experiments

#### Printeria Wet Lab

In regard to the lab work, several lines of experiments have been carried out with the objective of **ensuring the biological functioning of Printeria**. We have kept a record of our daily lab work through different **notebooks**, so you can access the documents for each group of experiments by clicking their respective buttons.

You can also read the collection of protocols we have followed to proceed with our experiments, so they could be easily repeated by future iGEM teams.

[Protocols](https://static.igem.org/mediawiki/2018/b/bc/T--Valencia_UPV--protocols.pdf)

**Level 0 assembly (DNA Basic parts adaptation)**

Printeria needs a toolkit of DNA basic parts adapted to be assembled with the [Golden Gate assembly method](https://2018.igem.org/Team%3AValencia_UPV/Design#GB). We have designed them as they appear on our [Design page](https://2018.igem.org/Team%3AValencia_UPV/Design). You can check the chronological construction phases of these parts going to:

* [Part Adaptation Notebook](https://static.igem.org/mediawiki/2018/d/d1/T--Valencia_UPV--Part_Adapt.pdf)
* [Our basic parts](https://2018.igem.org/Team%3AValencia_UPV/Part_Collection#bas)

**Destination vector experiments**

We have done several experiments to select the way to introduce the [Level 1 destination plasmid](https://2018.igem.org/Team%3AValencia_UPV/Design#GB), in order to obtain the lowest number of bacteria without the desired insert resulting from the Golden Gate assembly reaction, and thus trying to avoid the necessity of the plate screening step. The progressive process of decision has been recorded in our:

* [Destination vector experiments notebook](https://static.igem.org/mediawiki/2018/5/50/T--Valencia_UPV--Destination.pdf)

**Experiments with competent cells**

Several experiments were done to decide how to introduce the *E. coli* chassis into Printeria. It included transformation efficiency experiments with freeze drying competent cells and with competent cells maintained at -20ºC and room temperature. All the experiments are chronologically listed in our:

* [Experiments with competent cells notebook](https://static.igem.org/mediawiki/2018/7/77/T--Valencia_UPV--Competent.pdf)

**Level 1 assembly (Composite Parts Assembly)**

In order to characterize our DNA basic parts, we had to assemble composite parts using the Golden Gate technology ([Level 1 assembly](https://2018.igem.org/Team%3AValencia_UPV/Design#GB)) with our DNA basic part collection. By this way we were able to check the robustness of the protocol and to verify that its efficiency was high enough to be done by Printeria.

* [Composite Parts Assembly Notebook](https://static.igem.org/mediawiki/2018/5/50/T--Valencia_UPV--Composite.pdf)
* [Our Composite Parts](https://2018.igem.org/Team%3AValencia_UPV/Part_Collection#com)

**pBioBrickator plasmid construction and composite parts adaptation**

As it is explained in  [the Part Collection page](https://2018.igem.org/Team%3AValencia_UPV/Part_Collection) , we have designed the  [BBa\_K2656200 vector](http://parts.igem.org/Part%3ABBa_K2656200), adapted from the standard pSB1C3 backbone, to convert our Golden Gate transcriptional units to BioBricks. Thus, we were able to introduce each transcriptional unit into a BioBrick compatible vector with a Type IIS enzyme reaction. This process can be checked going to:

* [Composite part adaptation to BioBricks](https://static.igem.org/mediawiki/2018/8/85/T--Valencia_UPV--Biob.pdf)

#### Printeria device

In the Printeria project, the **experiments carried out on the machine** developed by the Printeria Hardware team stand out in importance. With them, we have tried to **verify the automation process** that our device offers to the user. Below are described both the materials and the procedures of the most important experiments our Printeria equipment.

**Input system**

In this experiment we wanted to test the feasibility of the concept of breaking the seal we put in pipette tips and blowing through the synge to get the contents out.

**Materials:**

+ Arduino
+ Peristaltic pump
+ Solenoid
+ Syringe
+ Input carrousel
+ Stepper motor
+ Sealed pipette tips

**Procedure:**

1. The pipette tips are loaded with 4
    μL of liquid and are placed in place on the input carrousel.
2. The stepper motor spins it to get the filled pipette tips under the syringe.
3. The solenoid is activated, moving down the syringe into the tip.
4. The pump blows filtered air into the tip, releasing the liquid.

**Digital microfluidics**

For the first experiment of droplet movement the objective was to determine the important parameters that affect this technology.

**Materials:**

+ HV supply
+ Pad prototype PCB 1
+ Parafilm
+ Silicone oil

**Procedure:**

1. A thin layer of silicone oil is placed on top of the pads of the PCB.
2. The parafilm is stretched to be as thin as possible.
3. Another layer of silicone oil is placed on top of the parafilm.
4. 15
    μL of water are placed on top of one of the pads.
5. We manually connected and disconnected wires from the HV voltage supply set at 300 V.

For the second iteration of droplet movement test the objective was to add automation to the movement:

**Materials:**

* HV supply
* Pad prototype PCB 2
* IC: HV507
* Arduino
* Parafilm
* Silicone oil

**Procedure:**

1. A thin layer of silicone oil is placed on top of the pads of the PCB.
2. The parafilm is stretched to be as thin as possible.
3. Another layer of silicone oil is placed on top of the parafilm.
4. 15
    μL of water are placed on top of one of the pads.
5. Using the arduino we send commands of which pad to turn on, moving the droplet

Extending onto the previous experiment we designed a PCB that would serve as the surface from printeria. This time we used 6 concatenated IC.

**Materials:**

* HV supply
* Pad prototype PCB final
* IC: HV507
* Arduino
* Parafilm
* Silicone oil

**Procedure:**

1. A thin layer of silicone oil is placed on top of the pads of the PCB.
2. The parafilm is stretched to be as thin as possible.
3. Another layer of silicone oil is placed on top of the parafilm.
4. 15
    μL of water are placed on top of one of the pads.
5. Using the arduino we send commands of which pad to turn on, moving the droplet.

**Hot/cold zone**

The objective of this experiment is to test the concept of a hot and cold zone to replace a thermocycler.

**Materials:**

+ Peltier device
+ Power resistor
+ Arduino
+ Variable voltage power supply
+ IRFZ44N mosfet
+ Thermistors
+ 2k2 and 10k resistors
+ Intel stock cooler
+ [BBa\_P10500 plasmid](http://parts.igem.org/Part%3ABBa_P10500)
+ gBlock of [BBa\_K2656024](http://parts.igem.org/Part%3ABBa_K2656024) adapted to be assembled into P10500
+ BsmBI endonuclease
+ T4 DNA ligase
+ Bovine serum albumine
+ T4 DNA ligase buffer
+ 10G electrocompetent cells
+ Electroporator
+ Electroporation cuvette
+ LB-agar plates with Kanamycin
+ SOC recovery medium

**Procedure:**

1. Assemble the circuit.
2. Set up the reaction following the Golden Gate assembly protocol.
3. Pipette the drop where the reaction is taking place between the zone at 16ºC and the one at 37ºC during [**25 cycles**](#imPrinteria) following these times:

| Temperature (ºC) | Time (minutes) |
| --- | --- |
| 37 | 3 |
| 16 | 4 |

###### Golden Gate assembly reaction protocol. That schema must be run 25 cycles.

4. Transform the electrocompetent cells with 9
    μL of the reaction result.

**Electroporator**

On this experiment the objective was to test the electroporator module for Printeria.

**Materials:**

+ Electroporator PCB
+ Arduino
+ HV Supply
+ Electroporation cuvette

**Procedure:**

To test the electroporation module, we transformed 10G electrocompetent cells with 1 to 2  μL of the [BBa\_K26560105](http://parts.igem.org/Part%3ABBa_K2656105) transcriptional unit previously assembled into an alpha GoldenBraid vector (KmR). Thus, this plasmid express the sfGFP protein and so express fluorescence under UV light.

1. Pipette 1  μL of DNA (80.5 ng/ μL) into 50  μL 10G aliquot
2. Transfer into the electroporation cuvette
3. Turn on the electroporation module, with 5 ms of set point and 1.5 kV.
4. Add 700  μL SOC liquid medium and incubate for 1h with vigorous shaking
5. Plate 300  μL into agar plate with kanamycin antibiotic
6. Incubate for 16-18h at 37ºC

### Parts characterization

This year, Valencia UPV iGEM team has designed an extensive [Part Collection](https://2018.igem.org/Team%3AValencia_UPV/Part_Collection) in purpose of allowing the user to design multiple genetic constructions and experiments. One of our main objectives has been to **show the user clear and structured information** about the pieces that make up the Printeria kit. For this reason, we have considered the characterization of the parts as a priority when developing the project. In this way, we have elaborated an **experimental protocol** which have allowed us to systematically obtain and structure information from the parts.

#### Experimental protocol

The Printeria Modeling and Lab team and have jointly designed an experimentation protocol for the laboratory experiments. Thanks to it, and from the colonies of the different TU the experimental data can be obtained, processed and ready to be optimized.

*Materials:*

* Printeria transcriptional units (see our Printeria [Part Collection](https://2018.igem.org/Team%3AValencia_UPV/Part_Collection))
* Measuring equipment: **Biotek Cytation3**
* **96 well plate**
* **MATLAB 2018a software**

*Protocol:*

1. A colony or a Glycerol stock corresponding to a TU assembled by our Lab team is used to inoculate LB culture medium (with the appropriate antibiotic, in the cases of the TU, with Kanamycin) and grow overnight at 37ºC and 250 r.p.m.
2. A 10:1 dilution of the culture is performed changing the culture media to M9 minimal medium and grow for 4 hours at 37ºC and 250 r.p.m.
3. The culture is chilled in an ice-water bath anfd the optical density at 600nm (OD600) of the culture is measured with a spectrophotometer. Then a calculation is performed (using this Excel sheet) to make a dilution in order to bring the OD600 of the culture to 0.1OD600 with the appropriate culture volume to inoculate 8 replicas into 200uL wells.
4. The experiment is designed on the measuring equipment. In our case, we use the Biotek Cytation3 equipment. We establish the equipment parameters.

   | Parameters | Description |
   | --- | --- |
   | Time | **06:00:00 (HH:MM:SS)** usually. **Measurement interval: 05:00 (MM:SS)** |
   | Number of samples | We normally set **8 samples of reporter protein for each TU colony** |
   | Number of medium samples | We normally set **8 samples of medium**. We normally use **LB or M9 medium with Kanamycin** |
   | Temperature | 37 ºC |
   | Shake | Double Orbital. Continuously. We shake the plate before each measure |
   | Absorbance. Optical Density (OD) measure | Wavelenght at **600 nm** emission |
   | Excitacion wavelength | We normally set **485 nm** for GFP or sfGFP and **500 nm** for YFP |
   | Emission wavelength | We normally set **528 nm** for GFP or sfGFP and **540 nm** for YFP |
   | Gain (G) | Normally the gain value is **G = 60**, although for proteins with lower fluorescence, it is recommended that G takes higher values. |

   ###### Biotek Cytation 3 experiment parameters
5. Samples are introduced into the 96 well plate and the experiment begins.
6. After the experiment, fluorescence and absorbance data obtained are exported to an Excel file.
7. We run the MATLAB [convert\_data.m](https://static.igem.org/mediawiki/2018/1/13/T--Valencia_UPV--convert_dataUPV2018.txt) script. This script uses several additional MATLAB files with which:
   1. We extract the fluorescence and absorbance matrices from the Excel file (see [readExperiment3.m](https://static.igem.org/mediawiki/2018/e/e4/T--Valencia_UPV--readExperiment3UPV2018.txt) MATLAB function).
   2. We apply the [corrections](#esquema_ops) to the data (see [plotExperiment\_yb.m](https://static.igem.org/mediawiki/2018/3/32/T--Valencia_UPV--plotExperiment_ybUPV2018.txt) MATLAB function).
   3. We save the data of FOD, OD, molecules and time in a **.mat format** file.

###### **Experiments changing RBS**

We have designed **two experiments** following the same [experimental protocol](#exp_protocol). In them we have assembled different Printeria TU with the same promoters, CDS ([sfGFP](http://parts.igem.org/Part%3ABBa_K2656013) reporter protein) and transcriptional terminator, but with **different RBS**. These experiments were used in our modeling to complete the characterization of the part in the Printeria Collection.

**Printeria TU**:

* **Strong expression**: [BBa\_K2656101](http://parts.igem.org/Part%3ABBa_K2656101).
* **Medium expression**: [BBa\_K2656103](http://parts.igem.org/Part%3ABBa_K2656103).
* **Weak expression**: [BBa\_K2656102](http://parts.igem.org/Part%3ABBa_K2656102).
* **Very weak expression**: [BBa\_K2656100](http://parts.igem.org/Part%3ABBa_K2656100), [BBa\_K2656104](http://parts.igem.org/Part%3ABBa_K2656104).

**Printeria RBS**:

* **Strong expression**: [BBa\_K2656009](http://parts.igem.org/Part%3ABBa_K2656009).
* **Medium expression**: [BBa\_K2656011](http://parts.igem.org/Part%3ABBa_K2656011).
* **Weak expression**: [BBa\_K2656010](http://parts.igem.org/Part%3ABBa_K2656010).
* **Very weak expression**: [BBa\_K2656008](http://parts.igem.org/Part%3ABBa_K2656008), [BBa\_K2656012](http://parts.igem.org/Part%3ABBa_K2656012).

[![](https://static.igem.org/mediawiki/2018/9/96/T--Valencia_UPV--optimization_RBS_exp1_RBS_graph_expUPV2018.png)](https://static.igem.org/mediawiki/2018/9/96/T--Valencia_UPV--optimization_RBS_exp1_RBS_graph_expUPV2018.png)

###### Experiment 1 changing RBS. Experimental data of strong, medium and weak protein expression RBS

[![](https://static.igem.org/mediawiki/2018/9/9c/T--Valencia_UPV--optimization_exp2_RBS_all_graph_expUPV2018.png)](https://static.igem.org/mediawiki/2018/9/9c/T--Valencia_UPV--optimization_exp2_RBS_all_graph_expUPV2018.png)

###### Experiment 2 changing RBS. Experimental data of strong and very weak protein expression RBS

[![](https://static.igem.org/mediawiki/2018/1/19/T--Valencia_UPV--optimization_exp2_RBS_2obj_graph_expUPV2018.png)](https://static.igem.org/mediawiki/2018/1/19/T--Valencia_UPV--optimization_exp2_RBS_2obj_graph_expUPV2018.png)

###### Experiment 2 changing RBS. Experimental data of very weak protein expression RBS

###### **Experiments changing promoters**

We have designed **an experiment** following the [experimental protocol](#exp_protocol). In them we have assembled different Printeria TU with the same RBS, CDS ([GFP](http://parts.igem.org/Part%3ABBa_K2656022) reporter protein) and transcriptional terminator, and with **different promoters**. After obtaining the results, and following the [optimization protocol](#optimization), we have obtained the parameters of the model and have validated our model.

**Printeria TU**:

* **Strong promoters**: [BBa\_K2656106](http://parts.igem.org/Part%3ABBa_K2656106)
* **Medium promoters**: [BBa\_K2656107](http://parts.igem.org/Part%3ABBa_K2656107)
* **Weak promoters**: [BBa\_K2656105](http://parts.igem.org/Part%3ABBa_K2656105)

**Printeria promoters**:

* **Strong promoters**: [BBa\_K2656005](http://parts.igem.org/Part%3ABBa_K2656005)
* **Medium promoters**: [BBa\_K2656007](http://parts.igem.org/Part%3ABBa_K2656007)
* **Weak promoters**: [BBa\_K2656004](http://parts.igem.org/Part%3ABBa_K2656004)

[![](https://static.igem.org/mediawiki/2018/0/07/T--Valencia_UPV--optimization_GFP_expUPV2018.png)](https://static.igem.org/mediawiki/2018/0/07/T--Valencia_UPV--optimization_GFP_expUPV2018.png)

###### Experiment 1 changing promoters. Experimental data of strong, medium and weak promoters.

###### **Experiment to compare reporter proteins with and without LVA tag**

In this experiment, that we have performed following the [experimental protocol](#exp_protocol), we have designed two TU. Both have the same promoters, RBS and transcriptional terminators, but **different CDS**. The CDS express the reporter protein **YFP**, however, one of them has a degradation LVA tag, which causes faster degradation.

**Printeria TU**:

* **YFP with LVA tag: [BBa\_K2656111](http://parts.igem.org/Part%3ABBa_K2656111)**
* **YFP without LVA tag: [BBa\_K2656112](http://parts.igem.org/Part%3ABBa_K2656112)**

**Printeria CDS**:

* **YFP with LVA tag: [BBa\_K2656020](http://parts.igem.org/Part%3ABBa_K2656020)**
* **YFP without LVA tag: [BBa\_K2656021](http://parts.igem.org/Part%3ABBa_K2656021)**

[![](https://static.igem.org/mediawiki/2018/9/91/T--Valencia_UPV--optimization_YFP_expUPV2018.png)](https://static.igem.org/mediawiki/2018/9/91/T--Valencia_UPV--optimization_YFP_expUPV2018.png)

###### Experiment 1 YFP vs. YFP LVA tag. Experimental data of YFP and YPF LVA tag reporter proteins.

###### **Comparative experiment between Golden Gate and BioBricks assembly method**

The comparison experiment aims to **compare the protein expression** of two genetic constructs obtained by the **Golden Gate** and **BioBricks assembly methods**. The [BBa\_K2656117](http://parts.igem.org/Part%3ABBa_K2656117) TU has been designed for this purpose, whose parts are identical to those constituting the BioBricks [BBa\_J364001](http://parts.igem.org/Part%3ABBa_J364001) construction. Both parts have been introduced in the same vector, and this one has been introduced in turn in the same strain **E. Coli 10G**. Finally, the fluorescence and absorbance of both constructions have been measured, following the usual [experimental protocol](#exp_protocol), and the following results have been obtained.

[![](https://static.igem.org/mediawiki/2018/1/17/T--Valencia_UPV--comparison_GGBB_graphUPV2018.png)](https://static.igem.org/mediawiki/2018/1/17/T--Valencia_UPV--comparison_GGBB_graphUPV2018.png)

### Reporter proteins comparisons

The reporter proteins are one of the essential elements in the experimentation process of the Valencia UPV iGEM team. Thanks to them, we can **quantify the protein expression** of our modified bacteria, and relate it to the [theoretical models designed](https://2018.igem.org/Team%3AValencia_UPV/Modeling#models). Proteins such as GFP, YFP, sfGFP, mRFP... are the proteins that provide us with information about the Printeria product, and that we want to show our user.

For this reason, the Printeria Modeling team and Web Lab have jointly elaborated a series of **experimental procedures** to obtain new information from the reporter proteins and use it in the treatment of experimental data.

* [Procedure for obtaining protein spectra](#spectra)
* [Comparison between sfGFP, GFP and YFP relative fluorescence intensity](#comparison)

#### Procedure for obtaining protein spectra

Obtaining **excitation and emission spectra** is a fundamental aspect in the process of a fluorescent protein characterization. **Each protein has a characteristic spectrum**, which indicates the emitted energy of the fluorescent molecule when it is excited at a certain wavelength.

The characterization of the reporter proteins by excitation and emission spectra are very importance during experimentation. When contrasting experimental information with theoretical results of our mathematical models, we experiment with numerous reporter proteins, such as fluorescence proteins or chromoproteins. However, the **fluorescence data obtained must be corrected by applying diferent operations** in order to obtain representative fluorescence data:

1. The subtraction of the medium fluorescence:
   ![](https://static.igem.org/mediawiki/2018/9/97/T--Valencia_UPV--fsubsUPV2018.png)

- The quotient of the fluorescence with the gain:
  ![](https://static.igem.org/mediawiki/2018/b/b0/T--Valencia_UPV--fgainUPV2018.png)
- The quotient of the fluorescence with excitation and emission efficiency:
  ![](https://static.igem.org/mediawiki/2018/2/21/T--Valencia_UPV--fcorrUPV2018.png)

[![](https://static.igem.org/mediawiki/2018/6/6e/T--Valencia_UPV--esquema_opsUPV2018.png)](https://static.igem.org/mediawiki/2018/6/6e/T--Valencia_UPV--esquema_opsUPV2018.png)

###### Correction process of fluorescence data

Until now, the corrections applied to the experiments were the subtraction of the fluorescence of the medium, and the division by the gain factor of the equipment. However, **the measured protein spectra can be normalized** with respect to the values that would have been obtained at the maximum excitation and emission wavelength.

Owing to this reason, **a protocol has been established** by the Lab and Modeling team **to obtain a more accurate spectrum of any reporter protein**.

*Materials:*

* Measuring equipment: **Biotek Cytation3**
* **96 well plate**
* **MATLAB 2018a software**

*Procedure:*

1. We look for the [theoretical spectra](https://www.thermofisher.com/es/es/home/life-science/cell-analysis/labeling-chemistry/fluorescence-spectraviewer.html) of the protein to be measured or similar molecules in order to determine the wavelength at which the protein is excited or emitted at maximum energy, i.e. where the theoretical spectral peaks occurs.
2. We define the protocol of our equipment to get the absorbance and fluorescence dataset. In our protocol, the most important parameters to be established are summarized in the following Table.

   | Parameters | Description |
   | --- | --- |
   | Number of samples | From **3 to 6 samples of reporter protein** and **3 samples of medium (LB or M9 medium)** |
   | Temperature | 37 ºC |
   | Shake | Double Orbital. 01:00 (MM:SS) |
   | Absorbance. Optical Density (OD) | Wavelenght at **600 nm** emission |
   | Excitacion and emission scans | The scans occur between **two wavelength limit values**. The established range will depend on the theoretical spectrum of the protein. |
   | Excitacion and emission wavelengths | These values will depend on the range, and therefore on the spectrum. Values far from the theoretical peak lead to more attenuated fluorescence curves, and values very close to the peak can lead to overlap and error in reading the data. Therefore, **a compromise must be reached between curve resolution and reading overlap**. |
   | Gain (G) | Normally the gain value is **G = 60**, although for proteins with lower fluorescence, it is recommended that G takes higher values. |

   ###### Experimental parameters
3. The experiment is introduced, and the experimental absorbance data and the fluorescence curves of the samples are obtained with the medium fluorescence correction applied.
4. The dataset is exported to an Excel file.
5. The MATLAB script [spectrum.m](https://static.igem.org/mediawiki/2018/f/f7/T--Valencia_UPV--spectrummUPV2018.txt) for fluorescent proteins is executed:
   1. Dataset is extracted from the Excel file. We discard readings that have suffered overlap, or that take negative values.
   2. The fluorescence curves of all samples are averaged, and the result is normalised (from 0 to 100%).
   3. Graphs of the normalized absorption and emission spectra are plotted. The X-axis represents the wavelength (nm), and the Y-axis represents the normalized fluorescence intensity (%).

In practical terms, the protocol has been applied to all of our reporter proteins: [GFP](http://parts.igem.org/Part%3ABBa_K2656022), [sfGFP](http://parts.igem.org/Part%3ABBa_K2656013), [YFP](http://parts.igem.org/Part%3ABBa_K2656021) and [mRFP](http://parts.igem.org/Part%3ABBa_K2656014). All results can be found in our parts collection as well as in the [iGEM catalog](http://parts.igem.org/Catalog).

[![](https://static.igem.org/mediawiki/2018/1/17/T--Valencia_UPV--GFP_spectrumUPV2018.png)](https://static.igem.org/mediawiki/2018/1/17/T--Valencia_UPV--GFP_spectrumUPV2018.png)

###### GFP spectra

[![](https://static.igem.org/mediawiki/2018/e/e8/T--Valencia_UPV--sfGFP_spectrumUPV2018.png)](https://static.igem.org/mediawiki/2018/e/e8/T--Valencia_UPV--sfGFP_spectrumUPV2018.png)

###### sfGFP spectra

[![](https://static.igem.org/mediawiki/2018/2/24/T--Valencia_UPV--YFP_spectrumUPV2018.png)](https://static.igem.org/mediawiki/2018/2/24/T--Valencia_UPV--YFP_spectrumUPV2018.png)

###### YFP spectra

[![](https://static.igem.org/mediawiki/2018/a/a8/T--Valencia_UPV--mRFP_spectrumUPV2018.png)](https://static.igem.org/mediawiki/2018/a/a8/T--Valencia_UPV--mRFP_spectrumUPV2018.png)

###### mRFP spectra

In the particular case of the reporter chromoprotein, such as [amilCP](http://parts.igem.org/Part%3ABBa_K2656018), we do not measure fluorescence, but absorbance. In this case, in order to obtain the corrected absorbance curve, we must subtract the absorbance of a medium with cells without chromoprotein from the cell absorbance data with the reporter protein. Once the data have been corrected, we normalized them in a range of 0 and 100. The figure below shows the blue cromoprotein spectra. The protocol used can be found in the MATLAB script [amilCP\_spectrum.m](https://static.igem.org/mediawiki/2018/8/8b/T--Valencia_UPV--amilCP_spectrumUPV2018.txt).

[![](https://static.igem.org/mediawiki/2018/2/2d/T--Valencia_UPV--amilCP_spectruM_graphUPV2018.png)](https://static.igem.org/mediawiki/2018/2/2d/T--Valencia_UPV--amilCP_spectruM_graphUPV2018.png)

###### amilCP spectra

Finally, we have also obtained the spectra of the fluorescein molecule. These spectra have been used to correct the fluorescence data used in the [Interlab Study](https://2018.igem.org/Team%3AValencia_UPV/InterLab) to obtain the Relative Fluorescence Units (RFU) to Molecules of Equivalent Fluorochrome (MEFL) conversion factor. In addition, the [comparison](#comparison) between GFP and sfGFP proteins RFU has been possible thanks to fluorescein spectra.

| Parameters | Value |
| --- | --- |
| Number of samples | 6 samples. 3 samples of medium (LB or M9 medium) |
| Temperature | 37 ºC |
| Shake | Double Orbital. 01:00 (MM:SS) |
| Absorbance. Optical Density (OD) | Wavelenght at **600 nm** emission |
| Emission range | [495 - 580] nm |
| Fixed excitation wavelength | 480 nm |
| Excitation range | [430 - 520] nm |
| Fixed emission wavelength | 545 nm |
| Gain (G) | 60 |

###### Experimental parameters

[![](https://static.igem.org/mediawiki/2018/1/11/T--Valencia_UPV--fluorescein_spectrumUPV2018.png)](https://static.igem.org/mediawiki/2018/1/11/T--Valencia_UPV--fluorescein_spectrumUPV2018.png)

###### Fluorescein spectra

#### Comparison between sfGFP, GFP and YFP relative fluorescence intensity

**Units of fluorescence data** are one of the main problems during processing the results of experiments in SynBio projects. Unlike absorbance, where there is a simple conversion between Optical Density (OD) and cell number, **there is no direct relationship between the Relative Units of Fluorescence (RFU) and the number of protein molecules in the cell**. Moreover, RFUs vary among reporter fluoroproteins: for example, an RFU of the GFP protein does not have to be equivalent to an RFU of the mRFP protein, sfGFP, etc.

Thanks to initiatives such as the [Interlab Study](https://2018.igem.org/Team%3AValencia_UPV/InterLab), we have been able to go a step further and obtain a **MEFL/cellGFP factor** for the equivalence between the RFU of the [GFP](http://parts.igem.org/Part%3ABBa_K2656022) protein and the Molecules of Equivalent Fluorochrome (MEFL). This relationship is an important breakthrough, as it can give us a more **accurate estimation** of the amount of GFP molecules in the cell.

However, **another reporter protein have been widely used in Printeria experiments, such as the [sfGFP](http://parts.igem.org/Part%3ABBa_K2656013)** or [YFP](http://parts.igem.org/Part%3ABBa_K2656021). In the case of **sfGFP**, these protein has a much faster folding than GFP, which translates into a **higher fluorescence intensity per molecule**. And that's really interesting in the field of experimentation. In the case of **YFP**, the Wet lab Printeria team has realized that **very little is known about this reporter protein**, so it has been proposed in this project to get as much information as possible about YFP and its variants (check [Printeria Improve](https://2018.igem.org/Team%3AValencia_UPV/Improve)). **But in both cases, the equivalence factor between fluorescence and equivalent molecules is unknown.**

Therefore, in order to obtain the MEFL/cellsfGFP and MEFL/cellYFP factors, the Printeria Modeling and Lab teams have designed a **comparative experiment between GFP and both reporter proteins**. The experiment consists of designing **two identical transcriptional units (TU), changing only the CDS sequence** so that each TU will produce GFP and our protein reporter (sfGFP or YFP), respectively. It should be added that this experiment is based on **two fundamental assumptions**:

1. The number of GFP molecules produced in the cells is equivalent to the number of MEFL.
2. Given two TU with identical promoters, RBS and terminators, but with different CDS, under the same experimental conditions, the number of molecules produced by each TU is the same.

Taking these axioms into account, the materials and procedure followed to calculate the MEFL/cell factors were as follows.

*Materials:*

* Measuring equipment: **Biotek Cytation3**
* **96 well plate**
* **MATLAB 2018a software**

*Procedure:*

1. From the fluorescein spectrum, and the fluorescence data obtained from the [Interlab Study experiment](https://2018.igem.org/Team%3AValencia_UPV/InterLab), we apply medium, gain and efficiency [corrections](#esquema_ops) .
2. The data are introduced in the Excel file of the Interlab Study. From the *Fluorescein standard curve* sheet we can obtain the MEFL/RFU factor, and then calculate the MEFL/cellGFP factor.
   [![](https://static.igem.org/mediawiki/2018/a/af/T--Valencia_UPV--calc_MEFLUPV2018.png)](https://static.igem.org/mediawiki/2018/a/af/T--Valencia_UPV--calc_MEFLUPV2018.png)

   | Parameters | Value |
   | --- | --- |
   | Culture volume (96 well plate) | 200  μL |
   | Cells per  μL per OD unit | 200 cells/OD  μ L |
   | MEFL/RFU factor | 4,38.1010 MEFL/RFU |
   | **MEFL/cellGFP factor** | **273.7 MEFL OD/RFU cell** |

   ###### MEFL/cell factor calculation
3. We establish the protocol of the experiment and the parameters of our equipment. The experiment consists in the measurement of the absorbance and fluorescence of **two TU with identical promoter, RBS and transcriptional terminator**, but whose CDS codifies for [GFP](http://parts.igem.org/Part%3ABBa_K2656105) and our reporter protein. We have done two differente experiments with the same protocol, by using [sfGFP TU](http://parts.igem.org/Part%3ABBa_K2656101) and [YFP TU](http://parts.igem.org/Part%3ABBa_K2656112). The most relevant information of the experiment is detailed in the following Table.

   | Parameters | Description |
   | --- | --- |
   | Time | 06:00:00 (HH:MM:SS). Measurement interval: 05:00 (MM:SS) |
   | Number of samples | 8 samples for each TU. Total samples: 16 |
   | Temperature | 37 ºC |
   | Shake | Double Orbital Continuous |
   | Absorbance. Optical Density (OD) | Wavelenght at **600 nm** emission |
   | Excitacion wavelength | 485 nm |
   | Emission wavelength | 528 nm |
   | Gain | 60 |

   ###### Experimental parameters
4. The experiment is introduced into the equipment and the absorbance and fluorescence curves are obtained, as well as the curve of **Fluorescence F/Absorbance OD** or **FOD ratio** with the applied medium fluorescence correction.
5. The absorbance and fluorescence data are exported to an Excel file.
6. Running the MATLAB [GFP\_sfGFP\_comparison.m](https://static.igem.org/mediawiki/2018/c/c9/T--Valencia_UPV--GPF_sfGFP_comparisonUPV2018.txt) and [GFP\_YFP\_comparison.m](https://static.igem.org/mediawiki/2018/b/be/T--Valencia_UPV--comparison_GFP_YFPUPV2018.txt) script (for each experiment):
   1. We extract the FOD data from the GFP and sfGFP from the Excel file.
   2. We apply the gain and efficiency [corrections](#esquema_ops)  of the spectrum to the FOD.
   3. We plot the FOD curves and look for a stationary equilibrium time interval in the expression of the GFP and sfGFP proteins. In our [experiment](#comp_graph), we have decided to chose the interval [145 - 290] min for sfGFP experiment and [40 - 350] min for YFP experiment.
   4. We obtain the average value of both regions.
   5. We calculate the number of GFP equivalent molecules or MEFL by multiplying the FOD data by the MEFL/cellGFP factor.
   6. If we assume that the number of molecules expressed by both TU in th experiment is the same, [the number of MEFL for the calculated GFP is the same as the number of MEFL for the other reporter protein](#cond_equiv_molec) (sfGFP or YFP).
   7. We calculate the MEFL/cellsfGFP and MEFL/cellYFP factors by dividing the MEFL number by the average FOD value of the sfGFP and YFP in the stationary region, respectively.

[![](https://static.igem.org/mediawiki/2018/a/aa/T--Valencia_UPV--comp_graph_sfGFPUPV2018.png)](https://static.igem.org/mediawiki/2018/a/aa/T--Valencia_UPV--comp_graph_sfGFPUPV2018.png)

###### Comparison between fluorescence intensity of GFP and sfGFP reporter proteins

[![](https://static.igem.org/mediawiki/2018/1/19/T--Valencia_UPV--comp_graph_YFPUPV2018.png)](https://static.igem.org/mediawiki/2018/1/19/T--Valencia_UPV--comp_graph_YFPUPV2018.png)

###### Comparison between fluorescence intensity of GFP and YFP reporter proteins

| Reporter protein | MEFL/cell factor (MEFL·OD/RFU·cell) |
| --- | --- |
| GFP | 273.7 |
| sfGFP | 121.4 |
| YFP | 57.3 |

###### MEFL/cell factors calculated applying comparison protocol

With MEFL/cell factors, **FOD data** obtained in any experiment in which GFP, sfGFP or YFP are used as reporter proteins **can be transformed into equivalent fluorescein molecules** by applying the following ratio:

[![](https://static.igem.org/mediawiki/2018/a/ad/T--Valencia_UPV--FOD_MEFL_conversionUPV2018.png)](https://static.igem.org/mediawiki/2018/a/ad/T--Valencia_UPV--FOD_MEFL_conversionUPV2018.png)

The new data give us more accurate estimation of the number of molecules in the cell. Consequently, by relating the experimental results with the theoretical mathematical models in the [optimization process](https://2018.igem.org/Team%3AValencia_UPV/Model#optimization), the **parameters of the model acquire values more consistent with their physical meaning. Also, we can work with equivalent molecules instead of using arbitrary units**.

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

# Team:Valencia UPV/Results

[![](https://static.igem.org/mediawiki/2018/8/8a/T--Valencia_UPV--pruebaPrinteriaWhiteUPV2018.png)](https://2018.igem.org/Team%3AValencia_UPV)

* Team

  + [Team](https://2018.igem.org/Team%3AValencia_UPV/Team)
  + [Attributions](https://2018.igem.org/Team%3AValencia_UPV/Attributions)
  + [Collaborations](https://2018.igem.org/Team%3AValencia_UPV/Collaborations)
  + [Gallery](https://2018.igem.org/Team%3AValencia_UPV/Gallery)
* Project

  + [Description](https://2018.igem.org/Team%3AValencia_UPV/Description)
  + [Design](https://2018.igem.org/Team%3AValencia_UPV/Design)
  + [Experiments](https://2018.igem.org/Team%3AValencia_UPV/Experiments)
  + [Inter Lab](https://2018.igem.org/Team%3AValencia_UPV/InterLab)
  + [Safety](https://2018.igem.org/Team%3AValencia_UPV/Safety)
  + [Improve](https://2018.igem.org/Team%3AValencia_UPV/Improve)
  + [Demonstrate](https://2018.igem.org/Team%3AValencia_UPV/Demonstrate)
  + [Modeling](https://2018.igem.org/Team%3AValencia_UPV/Model)
  + [Hardware](https://2018.igem.org/Team%3AValencia_UPV/Hardware)
  + [Software](https://2018.igem.org/Team%3AValencia_UPV/Software)
  + [Graphic Design](https://2018.igem.org/Team%3AValencia_UPV/GraphicDesign)
* Parts

  + [Parts](https://2018.igem.org/Team%3AValencia_UPV/Parts)
  + [Part Collection](https://2018.igem.org/Team%3AValencia_UPV/Part_Collection)
* Results

  + [Results](https://2018.igem.org/Team%3AValencia_UPV/Results)
  + [Demonstrate](https://2018.igem.org/Team%3AValencia_UPV/Demonstrate)
  + [Improve](https://2018.igem.org/Team%3AValencia_UPV/Improve)
* Human

  + [Human Practices](https://2018.igem.org/Team%3AValencia_UPV/Human_Practices)
  + [Integrated Human](https://2018.igem.org/Team%3AValencia_UPV/Human_Practices#Integrated)
  + [Public Engagement](https://2018.igem.org/Team%3AValencia_UPV/Public_Engagement)
* [Medals](https://2018.igem.org/Team%3AValencia_UPV/Medals)

[![](https://static.igem.org/mediawiki/2018/8/8a/T--Valencia_UPV--pruebaPrinteriaWhiteUPV2018.png)](https://2018.igem.org/Team%3AValencia_UPV)

[![](https://static.igem.org/mediawiki/2018/b/b9/T--Valencia_UPV--iconHardwareUPV2018.png)](https://2018.igem.org/Team%3AValencia_UPV/Hardware)
[![](https://static.igem.org/mediawiki/2018/d/d1/T--Valencia_UPV--iconSoftwareUPV2018.png)](https://2018.igem.org/Team%3AValencia_UPV/Software)[![](https://static.igem.org/mediawiki/2018/f/f6/T--Valencia_UPV--iconDesignUPV2018.png)](https://2018.igem.org/Team%3AValencia_UPV/Design)[![](https://static.igem.org/mediawiki/2018/0/0e/T--Valencia_UPV--iconModellingUPV2018.png)](https://2018.igem.org/Team%3AValencia_UPV/Model)

* Team

  + [Team](https://2018.igem.org/Team%3AValencia_UPV/Team)
  + [Attributions](https://2018.igem.org/Team%3AValencia_UPV/Attributions)
  + [Collaborations](https://2018.igem.org/Team%3AValencia_UPV/Collaborations)
  + [Gallery](https://2018.igem.org/Team%3AValencia_UPV/Gallery)
* Project

  + [Description](https://2018.igem.org/Team%3AValencia_UPV/Description)
  + [Design](https://2018.igem.org/Team%3AValencia_UPV/Design)
  + [Experiments](https://2018.igem.org/Team%3AValencia_UPV/Experiments)
  + [Model](https://2018.igem.org/Team%3AValencia_UPV/Model)
  + [Hardware](https://2018.igem.org/Team%3AValencia_UPV/Hardware)
  + [Software](https://2018.igem.org/Team%3AValencia_UPV/Software)
  + [Inter Lab](https://2018.igem.org/Team%3AValencia_UPV/InterLab)
  + [Safety](https://2018.igem.org/Team%3AValencia_UPV/Safety)
  + [Graphic Design](https://2018.igem.org/Team%3AValencia_UPV/GraphicDesign)
* Results

  + [Results](https://2018.igem.org/Team%3AValencia_UPV/Results)
  + [Improve](https://2018.igem.org/Team%3AValencia_UPV/Improve)
  + [Demonstrate](https://2018.igem.org/Team%3AValencia_UPV/Demonstrate)
* Parts

  + [Parts](https://2018.igem.org/Team%3AValencia_UPV/Parts)
  + [Part Collection](https://2018.igem.org/Team%3AValencia_UPV/Part_Collection)
* Human

  + [Human Practices](https://2018.igem.org/Team%3AValencia_UPV/Human_Practices)
  + [Integrated Human](https://2018.igem.org/Team%3AValencia_UPV/Human_Practices#Integrated)
  + [Public Engagement](https://2018.igem.org/Team%3AValencia_UPV/Public_Engagement)
* Awards

  + [Medals](https://2018.igem.org/Team%3AValencia_UPV/Medals)
  + [Awards](https://2018.igem.org/Team%3AValencia_UPV/Awards)
* [PRESS & CONTACT](https://2018.igem.org/Team%3AValencia_UPV/PressAndContact)

Stack Multipurpose HTML Template

![](https://static.igem.org/mediawiki/2018/thumb/7/7e/T--Valencia_UPV--ResultsTextoUPV2018.jpeg/1200px-T--Valencia_UPV--ResultsTextoUPV2018.jpeg.png)

#### Index

* [Goals](#Goals)
* [Results](#Results)
* [Considerations for replicating the project](#cons)
* [Future plans](#future)

### Goals

At the start of the Printeria project, we set themselves objectives in our respective areas in order to promote the automation of the cloning process and thus bring synthetic biology closer to people.

What we **wanted to achieve** above all, was to **miniaturize a high-tech laboratory** that was controlled by an **intuitive** and **pleasant** device. We also wanted the device to **show us the results before** we had them to avoid errors and save material and time. And at the present time, we are proud to say that we have done it.

#### Wet lab ambitions

|  |
| --- |
| Create a **standardized Golden Gate basic Parts Collection** to supply Printeria with the DNA toolkit to assemble composite parts. |
| Provide Printeria with the best way to transform competent bacteria with the highest transformation efficiency rate. |
| **Avoid** the **plate colony screening step** in order to eliminate the necessity of the researcher visual check, so easing Printeria automation. |

#### Hardware ambitions

|  |
| --- |
| **Integrate** multiple lab equipment around an automated friendly form. |
| Design an easy way of handling a big variety of input liquids providing a **straightforward integration** with Printeria. |
| Keep the **footprint** of the machine as small as possible. |
| Allow for **maximum flexibility** on the use of the machine. |

#### Software ambitions

|  |
| --- |
| Develop a **software** that allows the user to **control** the device. |
| Allow the user to **design a transcriptional unit** with the software. |
| Allow the user to **design multi-genetic constructs** with the software. |
| Get **non-professional** users to print basic genetic circuits. |
| Fill out a “recipe” repository with at least **20 recipes**. |
| Make a **user friendly interface** to interact with the software. |
| Control the device’s inventory through the software. |
| **Simulate** the bacterial growth and the protein expression. |

#### Modeling ambitions

|  |
| --- |
| Design simple mathematical models based which allows us to simulate the different Printeria genetic circuits. |
| **Optimize model parameters** to match simulation results to experimental data obtained from Printeria. |
| **Characterize the parts of our Part Collection** from the optimization results and provide the user with all the information about the Printeria kit. |
| Develop a **Simulation Tool** that allows the user to visualize a prediction of the results of their experiment before running it in Printeria. |
| Fill out a “recipe” repository with at least **20 recipes**. |
| Make a **user friendly interface** to interact with the software. |
| Control the device’s inventory through the software. |
| **Simulate** the bacterial growth and the protein expression. |

#### Human Practices ambitions

|  |
| --- |
| Try to show the **integration of feedback** from an **engineering point of view**, taking advantage of the fact that we are a multidisciplinary team. |
| Create **documents** that other **iGEM teams can use** in future projects. |
| Contact experts in both the field of art and the field of biotechnology. |

### Results

Once the objectives were clear we started to build Printeria, little by little. Once the project was finished, the conclusions reached after our  [experiments](https://2018.igem.org/Team%3AValencia_UPV/Experiments) were the following:

#### Wet Lab

* We have built a complete  [Part Collection](https://2018.igem.org/Team%3AValencia_UPV/Part_Collection)  of standardized Golden Gate basic parts. It is a fully functional DNA collection with extensive characterization of its parts, so providing the user with useful data to work with.

[![](https://static.igem.org/mediawiki/2018/a/a3/T--Valencia_UPV--Printone2UPV2018.jpeg)](https://static.igem.org/mediawiki/2018/a/a3/T--Valencia_UPV--Printone2UPV2018.jpeg)

###### **Image 1**. Some of our colours palette: **Printone**

* We found that the most suitable way to transform bacteria for Printeria is to electroporate electrocompetent cells previously stored at -20ºC and subsequent maintained at 4ºC during the assembly step.
* We proved that the assembly of composite parts using the destination vector in its linearized form significantly reduces the number of bacteria carrying the non-recombinant plasmid (false-positives colonies). However, in order to obtain a pure culture, it will always be necessary to go through the screening plate step.

  ![](https://static.igem.org/mediawiki/2018/0/0c/T--Valencia_UPV--BBa_K2656105_imgUPV2018.png)

  ###### **Image 2**. TU assembly of K2656105 with linearized plasmid

#### Hardware

* Laboratory equipment has been designed around **digital microfluidic technology**. Giving the machine a great degree of control over the processes that occur inside. This equipment includes a **thermal cycler** and an **electroporator**.

[![](https://static.igem.org/mediawiki/2018/e/eb/T--Valencia_UPV--ELECTROPORADOR1UPV2018.jpeg)](https://static.igem.org/mediawiki/2018/e/eb/T--Valencia_UPV--ELECTROPORADOR1UPV2018.jpeg)

[![](https://static.igem.org/mediawiki/2018/9/90/T--Valencia_UPV--PCBRESULTSUPV2018.jpeg)](https://static.igem.org/mediawiki/2018/9/90/T--Valencia_UPV--PCBRESULTSUPV2018.jpeg)

###### **Images 3-4.**Electroporator proof of concept and PCB final design.

* The entry system of Printeria allows for **quick placement of all the necessary consumables**. In addition, it can be further customized for any kind of liquid.

  [![](https://static.igem.org/mediawiki/2018/d/db/T--Valencia_UPV--ENTRADA5UPV2018.gif)](https://static.igem.org/mediawiki/2018/d/db/T--Valencia_UPV--ENTRADA5UPV2018.gif)

  ###### **Image 5.** Proof of concept of the entry system.
* Thanks to the use of Digital Microfluidics Printeria can produce **all the reactions in a small space**.
* All parts of the machine have been designed with **individual use in mind**.
* **Sensors** have been included to monitor bacterial growth.
* A **functional, economical and easy-to-use** solution has been achieved.

#### Software

* We developed **Printeria Controller**, a web application capable to **control all the functions** of our device.

  [![](https://static.igem.org/mediawiki/2018/4/45/T--Valencia_UPV--JobTabUPV2018.png)](https://static.igem.org/mediawiki/2018/4/45/T--Valencia_UPV--JobTabUPV2018.png)[![](https://static.igem.org/mediawiki/2018/6/64/T--Valencia_UPV--AdvancedTabUPV2018.png)](https://static.igem.org/mediawiki/2018/6/64/T--Valencia_UPV--AdvancedTabUPV2018.png)

  ###### **Image 6-7.** Printeria controller software interface
* Through the application it is possible to **select all the DNA parts needed** to design a transcriptional unit.
* We didn’t implement the multi-genetic circuit constructions because the team considered this construction will be part of the **future of printeria**.
* A non-experienced user can print a genetic circuit already created and stored in our recipe repository.

  [![](https://static.igem.org/mediawiki/2018/f/f5/T--Valencia_UPV--RecipesEasyTabUPV2018.png)](https://static.igem.org/mediawiki/2018/f/f5/T--Valencia_UPV--RecipesEasyTabUPV2018.png)[![](https://static.igem.org/mediawiki/2018/2/2e/T--Valencia_UPV--RecipesTabUPV2018.png)](https://static.igem.org/mediawiki/2018/2/2e/T--Valencia_UPV--RecipesTabUPV2018.png)

  ###### **Image 8-9.** Recipes and Parts interface of Printeria controller.
* We created a database with **27 recipes** assembled with our basic parts using the Golden Gate technology.
* The clean and explicative interface of our software allows both experienced and non-experienced users to navigate through all the options.
* In the **Inventory tab** of the software it is possible to know which **cartridges** have been **drained** and register when they are **filled**.

  [![](https://static.igem.org/mediawiki/2018/6/6b/T--Valencia_UPV--InventoryTabUPV2018.png)](https://static.igem.org/mediawiki/2018/6/6b/T--Valencia_UPV--InventoryTabUPV2018.png)

  ###### **Image 10.** Inventory interface of Printeria controller.
* With the modeling team we developed a simulation tool that allow us to predict the results.

#### Modeling

* **Three types of mathematical models** have been defined, with their scheme, reactions, parameters and equations: **constitutive model, inducible model pBAD/araC and inducible model LuxR-LuxI**.
* The **simulations** of the theoretical models have been executed and the analyses have been carried out **modifying the parameters** of the model in order to determine its influence.

  [![](https://static.igem.org/mediawiki/2018/f/f9/T--Valencia_UPV--mc_sim_analysis_graphUPV2018.png)](https://static.igem.org/mediawiki/2018/f/f9/T--Valencia_UPV--mc_sim_analysis_graphUPV2018.png)

  ###### **Image 11.** Constitutive expression model analysis. The graph represents de PoI stationary value as function of CR and p ratios. One example of how variable parameters influence the model.
* We have established a **protocol of experimentation** in the laboratory, a process of **correction and transformation** of the data, and a protocol of **multiobjective optimization** of parameters.
* **Multiobjective experiments** and optimization have been carried out satisfactorily with the constituent (changing promoters and RBS parts) and inducible LuxR-LuxI models and the optimal parameters of the parts have been estimated.
* The **spectra** of the different **reporter proteins** and the equivalence factors between fluorescence per unit of absorbance (FOD) and the equivalent molecules of fluorescein have been obtained in order to obtain a more exact and non-arbitrary estimation of the concentration of the protein inside the cell.
* Through experiments and the application of protocols, relevant information has been associated with the **registered parts**.
* The **Simulation Tool** has been developed in collaboration with Printeria Software team. That tool includes the mathematical models developed, and with it the user can predict the experimental results of a transcriptional unit just clicking a button.

### Considerations for replicating the project

* **Special care**: The electroporator has a capacitor at a really high voltage, act with care and avoid direct contact. You can isolate the PCB further with a layer of nail polish.
* Ensure that if you use **thermal paste** on the peltier devices and the power resistor that is a **non conductive paste.**
* For **3D Printed parts** we used the standar settings for PLA, 0.15mm layer height, 40% infill, 215Cº.
* The surface for the PCB is really hard to get right, give yourself some time to learn how to properly apply the coating.
* While preparing the **cables** for the connection with the **PCB** make sure that all of them make good contact.
* Remember that the experimental zone of the machine needs to be in **complete darkness**. We left the cover of that part transparent on the photos for display purposes.
* When **replicating Printeria biological function**, take into account that the pre-defined assembly protocol is originally conceibed to be use with Golden Gate standandardized basic DNA pieces. Thus, you can easily use the pieces of our  [Part Collection](https://2018.igem.org/Team%3AValencia_UPV/Part_Collection) , or you can adapt your desired pieces to the Golden Gate Technology.

#### Human Practices

* The **Kano model** has been the axis of the  [Integrated human](https://2018.igem.org/Team%3AValencia_UPV/Integrated_Human). With it: we have created a document that **other teams can use**, we have **classified** the feedback of our users and we have **taken advantage of social engagement**.
* We have made demonstrations in museums like the **MAG (Mustang Art Gallery)** and we have also agreed to make future in the creative space **Hangar** (Barcelona) and **El planetari de Castellón (Castellón de la plana)**.
* We have had the opportunity to **chat with experts** about biotechnology and art. We have shown them Printeria and they have offered us their most sincere **opinion** that in many cases has been **integrated in the design**.

### Considerations for replicating the project

* **Special care**: The electroporator has a capacitor at a really high voltage, act with care and avoid direct contact. You can isolate the PCB further with a layer of nail polish.
* Ensure that if you use **thermal paste** on the peltier devices and the power resistor that is a **non conductive paste.**
* For **3D Printed parts** we used the standar settings for PLA, 0.15mm layer height, 40% infill, 215Cº.
* The surface for the PCB is really hard to get right, give yourself some time to learn how to properly apply the coating.
* While preparing the **cables** for the connection with the **PCB** make sure that all of them make good contact.
* Remember that the experimental zone of the machine needs to be in **complete darkness**. We left the cover of that part transparent on the photos for display purposes.
* When **replicating Printeria biological function**, take into account that the pre-defined assembly protocol is originally conceibed to be use with Golden Gate standandardized basic DNA pieces. Thus, you can easily use the pieces of our  [Part Collection](https://2018.igem.org/Team%3AValencia_UPV/Part_Collection) , or you can adapt your desired pieces to the Golden Gate Technology.

### Future plans

Until now, we have achieved the construction of transcriptional units, which is the basic level of assembly for any genetic circuit. However, our future aim is to finally achieve the contruction of **multigenetical assemblies**  [(Level 2)](https://2018.igem.org/Team%3AValencia_UPV/Design#Level2), so increasing Printeria's application possibilities even more.

Moreover, we also want to reach the **parallelization** of several experiments. Currently, we are limited with the design of the input and output zones to only three reactions. Thus, in the future the modification of this design, expanding the carrousel and PCB, will allow us to do several more reactions at the same time.

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