iGEM 2019 — Team CU

[![](https://2019.igem.org/wiki/images/8/8b/HQ_page_logo.jpg)](https://2019.igem.org)

# Team:CU/Notebook

Medigo Blue, free responsive template

![](https://i.ibb.co/BwGVKgk/Logo2.png)CU iGEM

* [Home (current)](https://2019.igem.org/Team%3ACU)
* Our Project

  [Description and Inspiration](https://2019.igem.org/Team%3ACU/Description)
  [Design](https://2019.igem.org/Team%3ACU/Design)
  [Hardware](https://2019.igem.org/Team%3ACU/Hardware)
  [Modeling](https://2019.igem.org/Team%3ACU/Model)
* Team

  [Team Members](https://2019.igem.org/Team%3ACU/Team)
  [Attributions](https://2019.igem.org/Team%3ACU/Attributions)
  [Collaborations](https://2019.igem.org/Team%3ACU/Collaborations)
* [Safety](https://2019.igem.org/Team%3ACU/Safety)
* [Parts](https://2019.igem.org/Team%3ACU/Parts)
* Experiement

  [Experiement](https://2019.igem.org/Team%3ACU/Experiement)
  [Results](https://2019.igem.org/Team%3ACU/results)
  [Notebook](https://2019.igem.org/Team%3ACU/Notebook)
* Human Practices

  [About](https://2019.igem.org/Team%3ACU/Human_Practices)
  [Education and Puplic Engagement](https://2019.igem.org/Team%3ACU/Public_Engagement)
  [Integrated Human Practices](https://2019.igem.org/Team%3ACU/Integrated_Human_Practices)
* [Acknowledgement](https://2019.igem.org/Team%3ACU/acknowledgement)
* [Obstacles](https://2019.igem.org/Team%3ACU/Obstacles)

Medigo Blue, free responsive template

# NOTEBOOK

![](https://static.igem.org/mediawiki/2019/thumb/3/35/T--CU--Calender.png/1199px-T--CU--Calender.png)

# Experiments

Media Preparation

#01 Preparation of LB broth / LB Agar media :

## Background

Luria-Bertani (LB) broth is the most widely used medium for the growth of bacteria.

## Materials

All weights are per Liter optimize it according to your needs
Add Agarose only on the media that will be used for plates
Label the bottom of plates with antibiotic and date and seal with parafilm
Store the flasks containing LB media and the agar plates at 4°C.

## Protocol

**Estimated total time:** 60-70 Minutes

![](https://static.igem.org/mediawiki/2019/thumb/4/47/T--CU--Protocol1.png/857px-T--CU--Protocol1.png)

#02 Preparation of SOC Media

## Background

SOC media (Super Optimal Broth with Catabolic repressor) for competent cell test and bacterial transformation.

## Materials

Prepare the following for 1L SOC media

* 250mM KCL by dissolving 1.86g in 100mL dH2O and take from solution 10mL
* 2M MgCl2 by dissolving 19g in 90mL dH2O and take 5mL
* 1M Glucose by dissolving 18g in 90mL dH2O and take 20mL

![](https://static.igem.org/mediawiki/2019/thumb/8/81/T--CU--SOC_Media.png/1200px-T--CU--SOC_Media.png)

## Protocol

**Estimated total time:** 120 Minutes

![](https://static.igem.org/mediawiki/2019/f/fc/T--CU--pro3.png)

Competent cell preparation

## Background

The prerequisite for bacteria to undergo transformation is its ability to take up free, extracellular genetic material. Competent cells are more likely to incorporate foreign DNA if their cell walls are altered so that DNA can pass through more easily. Cells are made competent by a process that uses calcium chloride and heat shock.

## Materials

* Once you enter the lab open the Ice maker to chill the falcons and cryovials
* Be sure that the solutions are prepared and put them on ice ALL THE TIME
* Bring the Material altogether ,Clean the bench and start working

![](https://static.igem.org/mediawiki/2019/thumb/2/2a/T--CU--competent_cells.png/1200px-T--CU--competent_cells.png)

## Protocol

**Estimated total time:** 240 Minutes (4 hrs)

![](https://static.igem.org/mediawiki/2019/thumb/4/4c/T--CU--pro4.png/1200px-T--CU--pro4.png)
![](https://static.igem.org/mediawiki/2019/thumb/f/f7/T--CU--pro5.png/1172px-T--CU--pro5.png)

Transformation

## Background

Transformations are essential to using the DNA Distribution Kits: resuspend the DNA sample in a well, transform the DNA into competent cells, and select single colonies.
When transforming competent cells, both timing and temperature are very important. Use a lab timer, follow the incubation temperatures closely, and keep materials on ice when required.

## Materials

* First thing to do is to pre-chill the eppendorfs , make sure the ice maker is working
* Adjust the water bath to 42 THIS IS CRITICAL
* Pre-warm the SOC and the plates before use

![](https://static.igem.org/mediawiki/2019/thumb/7/7f/T--CU--Transformation.png/1200px-T--CU--Transformation.png)

## Protocol

**Estimated total time:** 2 hours (plus 14-18 hour incubation)

![](https://static.igem.org/mediawiki/2019/thumb/3/31/T--CU--PRO6.png/1200px-T--CU--PRO6.png)
![](https://static.igem.org/mediawiki/2019/thumb/8/8b/T--CU--PRO7.png/1199px-T--CU--PRO7.png)
![](https://static.igem.org/mediawiki/2019/thumb/d/df/T--CU--PRO8.png/1200px-T--CU--PRO8.png)

We tried this protocol several times and performed changes in the steps like:

* Using different nourishment media such as SOC and LB
* Using different volumes of those nourishment media (200 µl, 950µl)
* Performing different incubation time (45 mins, 1 hr, 2 hrs), and also
  heat shock time (40 sec, 45 sec, 1 min, 1.5 mins)
  But unfortunately we couldn’t get single transformed cells.

Plasmid miniprep

Fast-n-Easy Plasmid Mini-Prep Kit (jena bioscience)

## Materials

## Reagents

* Add 12 ml Ethanol(96-99%) to washing buffer.
* add 0.8 mgRNase A to neutralization buffer.

![](https://static.igem.org/mediawiki/2019/f/f9/T--CU--unnamed.png)
![](https://static.igem.org/mediawiki/2019/2/23/T--CU--pasted_image_0.png)
![](https://static.igem.org/mediawiki/2019/thumb/e/e5/T--CU--PRO9_%281%29.png/1055px-T--CU--PRO9_%281%29.png)
![](https://static.igem.org/mediawiki/2019/9/99/T--CU--PRO10.png)

PureYield™ Plasmid Miniprep System

Before lysing cells and purifying DNA, prepare the Column Wash Solution by adding ethanol. Cap tightly after addition. 100 mL of 95% ethanol.

## Materials

## Reagents

* 27ml Cell Lysis Buffer (CLC) (Blue)
* 90ml Neutralization Solution (NSC)
* 2 × 27ml Endotoxin Removal Wash (ERB)
* 25ml Column Wash Solution (CWC)
* 10ml Elution Buffer (EBB)
* 250 PureYield™ Minicolumns
* 250 PureYield™ Collection Tubes

![](https://static.igem.org/mediawiki/2019/5/5d/T--CU--pasted_image_0_%281%29.png)

## Protocol

**Estimated total time:**1 hr

![](https://static.igem.org/mediawiki/2019/thumb/f/f4/T--CU--pro11.png/1199px-T--CU--pro11.png)
![](https://static.igem.org/mediawiki/2019/thumb/4/4b/T--CU--PRO12.png/1200px-T--CU--PRO12.png)
![](https://static.igem.org/mediawiki/2019/thumb/3/3a/T--CU--PRO13.png/1200px-T--CU--PRO13.png)

Agarose gel (1%)

## Background

Agarose concentrations mainly depend on the length of the loaded DNA.

## Materials

## Reagents

1x TAE buffer

![](https://static.igem.org/mediawiki/2019/thumb/e/e5/T--CU--pro14_%281%29.png/1200px-T--CU--pro14_%281%29.png)

## Protocol

![](https://static.igem.org/mediawiki/2019/thumb/2/27/T--CU--pro14_%282%29.png/1198px-T--CU--pro14_%282%29.png)
![](https://static.igem.org/mediawiki/2019/thumb/b/bc/T--CU--pro14_%283%29.png/1199px-T--CU--pro14_%283%29.png)

Restriction digestion

We digested inserts and vectors (pGEX-4T-1) using EcoRI and PstI (Thermo Scientific)
First, gBlocks (inserts) were resuspended in nuclease-free water following these steps
Before opening the tube, spin it down in a microcentrifuge for 3–5 seconds to ensure the DNA is in the bottom of the tube. The pellet can become statically charged and, without this step, can either fly out of the tube or remain in the cap, resulting in loss of yield.

1. Add molecular grade water, or a buffer such as IDTE, to reach a final concentration of 10 ng/µL. Our experiments have shown that storage concentrations <1 ng/µL result in loss of material due to adherence to the plastic tube in the absence of a carrier such as tRNA.
2. Vortex briefly.
3. Incubate at approximately 50°C for 15–20 min.
   Heating the tube will ensure the solvent comes in contact with the tiny pellet, even if it is stuck to the side of the tube. Thus, this step will increase the likelihood that the entire pellet will be resuspended.
4. Briefly vortex and centrifuge.

gBlocks were resuspended in 20 µL only to reach final concentration 50 ng/µL.

**Buffer:** O buffer (Orange) is the most suitable buffer for both enzymes.

**Buffer working conc. :**  1X

**Incubation temperature:**  37°C

**Incubation time:**  1-16 hours (we tried 1 hr which resulted in partial digestion, but both 6 hr and overnight were appropriate incubation times for complete digestion)

![](https://static.igem.org/mediawiki/2019/thumb/b/bb/T--CU--proo.png/1198px-T--CU--proo.png)

PstI **is not inactivated** by incubation at 80°C for 20 min.
There are many options to deactivate PstI:

1. Extract with phenol/chloroform.
2. Use PCR-Purification kit
3. Load directly to the gel electrophoresis.

Ligation

We ligated inserts and vectors using T4 DNA Ligase (Jena Bioscience)

There are 2 buffers that can be used in this ligation reaction:

## Standard Ligation Buffer, 10x conc.

500 mM Tris-HCl pH 7.8 at 25 °C, 100 mM MgCl2, 100 mM DTT, 10 mM ATP and 25 µg/ml BSA

## Fast Ligation Buffer, 2x conc.

60 mM Tris-HCl pH 7.8 at 25 °C, 20 mM MgCl2, 20 mM DTT, 2 mM ATP and 10 % PEG

We went through the fast ligation reaction, so we performed the following steps:

![](https://static.igem.org/mediawiki/2019/thumb/c/cd/T--CU--pto55.png/1200px-T--CU--pto55.png)

Then incubate for 1 hr at 23 °C.

Cell free expression of proteins

## Background

Production of proteins with Cell-Free kit from Arbor Biosciences.

## Protocol

**Estimated total time:**30 Minutes without incubation time

![](https://static.igem.org/mediawiki/2019/thumb/0/0f/T--CU--pro56_%281%29.png/932px-T--CU--pro56_%281%29.png)
![](https://static.igem.org/mediawiki/2019/thumb/8/86/T--CU--pro56_%282%29.png/1200px-T--CU--pro56_%282%29.png)

YPD/ASW Media

## Subtitle

YPD media with artificial seawater for preparing plates

## Background

YEPD or yeast extract peptone dextrose, also often abbreviated as YPD.
YPD medium is a complex medium often used for growing S. cerevisiae. It is composed of yeast extract (for vitamins and other nutrients), peptone (broken down proteins), and dextrose (the energy source for the yeast). After the solution is prepared, it is autoclaved so that it is sterile when used.

## Materials

## Reagents

![](https://static.igem.org/mediawiki/2019/thumb/b/b3/T--CU--pro101_%282%29.png/1200px-T--CU--pro101_%282%29.png)
![](https://static.igem.org/mediawiki/2019/thumb/4/4d/T--CU--pro101_%283%29.png/120px-T--CU--pro101_%283%29.png)

## Protocol

**Estimated total time:**30 Minutes without incubation time

![](https://static.igem.org/mediawiki/2019/thumb/0/0f/T--CU--pro56_%281%29.png/932px-T--CU--pro56_%281%29.png)
![](https://static.igem.org/mediawiki/2019/thumb/8/86/T--CU--pro56_%282%29.png/1200px-T--CU--pro56_%282%29.png)

Plasmid miniprep

Fast-n-Easy Plasmid Mini-Prep Kit (jena bioscience)

## Materials

## Reagents

* Add 12 ml Ethanol(96-99%) to washing buffer.
* add 0.8 mgRNase A to neutralization buffer.

![](https://static.igem.org/mediawiki/2019/f/f9/T--CU--unnamed.png)
![](https://static.igem.org/mediawiki/2019/2/23/T--CU--pasted_image_0.png)
![](https://static.igem.org/mediawiki/2019/thumb/e/e5/T--CU--PRO9_%281%29.png/1055px-T--CU--PRO9_%281%29.png)
![](https://static.igem.org/mediawiki/2019/9/99/T--CU--PRO10.png)

PureYield™ Plasmid Miniprep System

Before lysing cells and purifying DNA, prepare the Column Wash Solution by adding ethanol. Cap tightly after addition. 100 mL of 95% ethanol.

## Materials

## Reagents

* 27ml Cell Lysis Buffer (CLC) (Blue)
* 90ml Neutralization Solution (NSC)
* 2 × 27ml Endotoxin Removal Wash (ERB)
* 25ml Column Wash Solution (CWC)
* 10ml Elution Buffer (EBB)
* 250 PureYield™ Minicolumns
* 250 PureYield™ Collection Tubes

![](https://static.igem.org/mediawiki/2019/5/5d/T--CU--pasted_image_0_%281%29.png)

## Protocol

**Estimated total time:**1 hr

![](https://static.igem.org/mediawiki/2019/thumb/f/f4/T--CU--pro11.png/1199px-T--CU--pro11.png)
![](https://static.igem.org/mediawiki/2019/thumb/4/4b/T--CU--PRO12.png/1200px-T--CU--PRO12.png)
![](https://static.igem.org/mediawiki/2019/thumb/3/3a/T--CU--PRO13.png/1200px-T--CU--PRO13.png)


[![](https://2019.igem.org/wiki/images/8/8b/HQ_page_logo.jpg)](https://2019.igem.org)

# Team:CU/Experiments

Medigo Blue, free responsive template

![](https://i.ibb.co/BwGVKgk/Logo2.png)CU iGEM

* [Home (current)](https://2019.igem.org/Team%3ACU)
* Our Project

  [Description and Inspiration](https://2019.igem.org/Team%3ACU/Description)
  [Design](https://2019.igem.org/Team%3ACU/Design)
  [Hardware](https://2019.igem.org/Team%3ACU/Hardware)
  [Modeling](https://2019.igem.org/Team%3ACU/Model)
* Team

  [Team Members](https://2019.igem.org/Team%3ACU/Team)
  [Attributions](https://2019.igem.org/Team%3ACU/Attributions)
  [Collaborations](https://2019.igem.org/Team%3ACU/Collaborations)
* [Safety](https://2019.igem.org/Team%3ACU/Safety)
* [Parts](https://2019.igem.org/Team%3ACU/Parts)
* Experiement

  [Experiement](https://2019.igem.org/Team%3ACU/Experiement)
  [Results](https://2019.igem.org/Team%3ACU/results)
  [Notebook](https://2019.igem.org/Team%3ACU/Notebook)
* Human Practices

  [About](https://2019.igem.org/Team%3ACU/Human_Practices)
  [Education and Puplic Engagement](https://2019.igem.org/Team%3ACU/Public_Engagement)
  [Integrated Human Practices](https://2019.igem.org/Team%3ACU/Integrated_Human_Practices)
* [Acknowledgement](https://2019.igem.org/Team%3ACU/acknowledgement)
* [Obstacles](https://2019.igem.org/Team%3ACU/Obstacles)

# Experiments

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

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

### What should this page contain?

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

### Inspiration

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

[![](https://2019.igem.org/wiki/images/8/8b/HQ_page_logo.jpg)](https://2019.igem.org)

# Team:CU/Results

Medigo Blue, free responsive template

![](https://i.ibb.co/BwGVKgk/Logo2.png)CU iGEM

* [Home (current)](https://2019.igem.org/Team%3ACU)
* Our Project

  [Description and Inspiration](https://2019.igem.org/Team%3ACU/Description)
  [Design](https://2019.igem.org/Team%3ACU/Design)
  [Hardware](https://2019.igem.org/Team%3ACU/Hardware)
  [Modeling](https://2019.igem.org/Team%3ACU/Model)
* Team

  [Team Members](https://2019.igem.org/Team%3ACU/Team)
  [Attributions](https://2019.igem.org/Team%3ACU/Attributions)
  [Collaborations](https://2019.igem.org/Team%3ACU/Collaborations)
* [Safety](https://2019.igem.org/Team%3ACU/Safety)
* [Parts](https://2019.igem.org/Team%3ACU/Parts)
* Experiement

  [Experiement](https://2019.igem.org/Team%3ACU/Experiement)
  [Results](https://2019.igem.org/Team%3ACU/results)
  [Notebook](https://2019.igem.org/Team%3ACU/Notebook)
* Human Practices

  [About](https://2019.igem.org/Team%3ACU/Human_Practices)
  [Education and Puplic Engagement](https://2019.igem.org/Team%3ACU/Public_Engagement)
  [Integrated Human Practices](https://2019.igem.org/Team%3ACU/Integrated_Human_Practices)
* [Acknowledgement](https://2019.igem.org/Team%3ACU/acknowledgement)
* [Obstacles](https://2019.igem.org/Team%3ACU/Obstacles)

# Results

Here you can describe the results of your project and your future plans.

### What should this page contain?

* Clearly and objectively describe the results of your work.
* Future plans for the project.
* Considerations for replicating the experiments.

### Describe what your results mean

* Interpretation of the results obtained during your project. Don't just show a plot/figure/graph/other, tell us what you think the data means. This is an important part of your project that the judges will look for.
* Show data, but remember **all measurement and characterization data must also be on the part's Main Page on the Registry.** Otherwise these data will not be in consideration for any medals or part awards!
* Consider including an analysis summary section to discuss what your results mean. Judges like to read what you think your data means, beyond all the data you have acquired during your project.

### Project Achievements

You can also include a list of bullet points (and links) of the successes and failures you have had over your summer. It is a quick reference page for the judges to see what you achieved during your summer.

* A list of linked bullet points of the successful results during your project
* A list of linked bullet points of the unsuccessful results during your project. This is about being scientifically honest. If you worked on an area for a long time with no success, tell us so we know where you put your effort.

### Inspiration

See how other teams presented their results.

* [2014 TU Darmstadt](https://2014.igem.org/Team%3ATU_Darmstadt/Results/Pathway)
* [2014 Imperial](https://2014.igem.org/Team%3AImperial/Results)
* [2014 Paris Bettencourt](https://2014.igem.org/Team%3AParis_Bettencourt/Results)