Team:BGU Israel/Experiments
Experiments
Tech
Calcium Phosphate Transfection
Title: Transfection to microglia and astrocytes via “calcium phosphate” method.
Conducted by: Sagi Angel
Date: 3-7.6.18
Aim: In this experiment we have tried to use the calcium phosphate protocol in order to transfect microglia astrocytes and HEK as control in GFP gene. The use of HEK cells as control, is due to its ability to be transfected relatively easily by the various techniques, including calcium phosphate method.
Importance: This experiment was carried out in parallel with experiments using different methods of transfection (different reagents and electroporation) in order to find an efficient way of inserting our plasmids into astrocytes and microglia for the continuation of the project.
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Calcium Phosphate
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Theoretical background: Transfection of DNA into cells via calcium phosphate is a simple, efficient and inexpensive method is to transfect eukaryotic cells via calcium phosphate co-precipitation with DNA (Graham and van der Eb, 1973). The insoluble calcium phosphate precipitate with the attached DNA adheres to the cell surface and is brought into the cells by endocytosis. Calcium phosphate transfection has been optimized and widely used with many adherent and non-adherent cell lines (Jordan et al., 1996). Calcium phosphate transfection can result in transient expression of the delivered DNA in the target cell, or establishment of stable cell lines.
Procedure:
The ingredients prepared according to the protocol with the GFP gene plasmid:
- 23 ul of PUC GFP DNA ,187 ul OF 1M CaCl2, DDW up to 750ul + 750 HEBSX2
- All ingredients were made and then filtered in 0.22 filter for sterile solution
- The ingredients were mixed for 30 minutes in a 1.5 ml Eppendorf for the 6 wells plate (250ul of complete reagent for each well)
- In the 6 wells there were 2 options for the transformation:
a) Medium removed, 250ul reagent added. after 30 min new medium(2.5ml) added
b) 250ul reagent added + old medium(2.5ml)
- After 8 hours all old medium removed and added new 2.5ml of relevantmedium
Design:
[ADD PHOTO OR TABLE]
References:
- Chen, Y., Lu, B., Yang, Q., Fearns, C., Yates, J. R., 3rd and Lee, J. D. (2009). Combined integrin phosphoproteomic analyses and small interfering RNA--based functional screening identify key regulators for cancer cell adhesion and migration. Cancer Res 69(8): 3713-3720.
- Graham, F. L. and van der Eb, A. J. (1973). A new technique for the assay of infectivity of human adenovirus 5 DNA. Virology 52(2): 456-67.
- Jordan, M., Schallhorn, A. and Wurm, F. M. (1996). Transfecting mammalian cells: optimization of critical parameters affecting calcium-phosphate precipitate formation. Nucleic Acids Res 24(4): 596-601.
Tec-Monterrey
Purpose of collaboration:
Provide data about the chemical makeup of water sources in Israel, present projects and receive feedback.
Description:
The collaboration with the Tec-Monterrey team was initiated through Instagram.We set up a skype meeting to meet, discuss our projects, and help each other.
On August 15th 2018, we participated in a skype talk and learned that the Tec-Monterrey team is focused on water pollutant and collecting data regarding the chemical makeup of water sources in different countries. We were glad to help and to assist the team with data collection form Israel. We contacted Professor Ovadia Lev, whose research focuses on identifying organic-sulfur compounds in water pollutants and got the required data for Tec-Monterrey team.
Through this collaboration, we learned about our local water sources in Israel. We found that in Israel pollutants in drinking water is very rare and that most of the water in Israel is recycled. Most of the water from plants and sewage is recycled in water treatment plants that is being used for agriculture. We learned that by testing various micro-pollutants and determine their levels, the recycled water will be used (or not) for agriculture.We sent a concentration of the findings obtained in extensive surveys conducted in partnership with the Water Authority laboratory.
We hope this information had a meaningful impact for Tec-Monterrey’s project!
Duesseldorf
Purpose of collaboration:
Bring the topic of Synthetic Biology close to the public
Description:
Previous iGEM teams from the Heinrich-Heine university of Düsseldorf created a tradition of bringing Synthetic Biology to the public eye by sharing themed postcards.
Every year, many iGEM teams join this campaign.
This year, we designed a postcard and sent it to the Düsseldorf team for distribution.
We were inspired by this project and printed extra copies of our postcard that are posted in our human practice events.
US_AFRL_CarrollHS
Purpose of collaboration:
Increase exposure for our team on social media.
Description:
We happily took part in a marketing project spearheaded by US_AFRL_CarrollHS called “Mike the Microbe".
We created caricatures of ourselves with the Mike the Microbe character and posted them on Instagram.
This lead to further exposure to other iGEM teams and was a lot of fun!
MichiganState
Purpose of collaboration:
Present projects and receive feedback.
Description:
The group contacted us through Instagram, and in fact was the first group we talked to through social media. We decided to organize a Skype call in which we presented our projects to each other. It was very interesting to meet and discuss our project with another group and we enjoyed getting to know the team members from the University of Michigan.