Difference between revisions of "Team:BGU Israel/Experiments"

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       <h1>HebrewU / HujiGEM</h1>
 
       <h1>HebrewU / HujiGEM</h1>
  
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       <p class="justified">
+
       <p align="center">
−
<u>Purpose of collaboration:</u><br/>
+
    Calcium Phosphate Transfection<strong><u></u></strong>
−
We collaborated with HebrewU in lab work and by hosting the team for a mini-jamboree.<br/>
+
</p>
−
<br/>
+
<p>
−
<u>Description:</u><br/>
+
    <strong>
−
In addition to our lab work, it was important for our team to contact other teams in the iGEM community.  Our team initially participated in the Synthetic Biology Conference at the Weizmann institute that inspire us to use and develop synthetic biology tools to address unsolved problems. Furthermore, a special session was devoted to the iGEM and we had the chance to hear and talk with previous iGEM team members and judges. We were very excited and encouraged about the achievements of previous iGEM teams. At this conference, we also met the HeberwU iGEM team the additional Israeli team participating in the 2018 iGEM contest. As the HeberwU is the first iGEM team of the Hebrew University in Jerusalem that was initiated by students, we reached out thier team members and help them how to build a iGEM team based on our university's iGEM experience and resources. From this point on, we had a constant and efficient collaboration with the HebrewU team.
+
        <u>Title</u>
−
 
+
        :
−
<ol>
+
        <br/>
−
<li>We provided reagents, advised on how to organize the team, where to look for funding, and how to prioritize tasks according to the given timetable.  For example, the HebrewU team was unable to complete the Interlab task, as they were missing competent cells for the necessary transformations. We invited the HebrewU team to our lab and a team member was hosted by our team. We taught him\her the relevant protocol, and gave several Eppendorfs filled with competent cells Dh5a, so they were able to complete their Interlab task.</li>
+
    </strong>
−
 
+
    Transfection to microglia and astrocytes via “calcium phosphate” method
−
<li>HebrewU organized a wonderful Safety Poem competition, to which we gladly added our submission:</li>
+
    <br/>
−
<blockquote cite="Sagi Angel / OriginALS team member">
+
    <u>
−
<p>This is not a song about safety, this is a song about love!<br/>
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        <br/>
−
It's all started when you first wore your gloves<br/>
+
        <strong>Conducted by</strong>
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Small, nitrile, in the color of blue<br/>
+
    </u>
−
That’s when I realized - I love you<br/>
+
    <strong>
−
The way that you said 'always use goggles'<br/>
+
        :
−
Like warm LB my heart turned into a puddle<br/>
+
        <br/>
−
It's kinda funny how you read the MSDS<br/>
+
    </strong>
−
While I didn’t knew what to do with broken glass<br/>
+
    Sagi
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You kept your bench so sterile and clean</br>
+
</p>
−
When its comes to fire safety-you are the queen<br/>
+
<p>
−
If I was drowning in lab work you kept me afloat<br/>
+
    <strong>
−
So why did you leave and hung your white coat</p>
+
        <u>Date</u>
−
</blockquote>
+
        :
−
 
+
        <br/>
−
<li>On September 3th 2018, we hosted the HebrewU team for a mini-Jamboree at Ben-Gurion University. During this meetup, each team presented their project and received feedbacks regarding their scientific questions and  presentation improvements and discussed unsolved  problem. The questions asked by the team members at this meetup allowed us to evaluate our project and to raise project's aspects that were need to be improved. We ended this meetup with an outdoor activity that allow us to get know each other and to have a have a short break from lab work.</li>
+
    </strong>
−
 
+
    3-7.6.18
−
<li>Finally, we were humbled and honored that the HebrewU iGEM team decided to acknowledge our team as their project mentors. We truly believe that scientific collaboration is the way to advance research, as it is being encouraged in this student competition and that we will embrace in our future careers!</li>
+
</p>
 +
<p>
 +
    <strong><u>Aim:</u></strong>
 +
</p>
 +
<p>
 +
    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.
 +
</p>
 +
<p>
 +
    <strong><u>Importance:</u></strong>
 +
</p>
 +
<p>
 +
    This experiment was carried out in parallel with experiments using
 +
    different methods of transfection (different reagents and electrophoresis)
 +
    in order to find an efficient way of inserting our plasmids into astrocytes
 +
    and microglia for the continuation of the project.
 +
</p>
 +
<p>
 +
    <u></u>
 +
</p>
 +
<table border="1" cellspacing="0" cellpadding="0">
 +
    <tbody>
 +
        <tr>
 +
            <td width="208" valign="top">
 +
                <p>
 +
                    Experiments
 +
                </p>
 +
            </td>
 +
            <td width="208" valign="top">
 +
                <p>
 +
                    Protocols
 +
                </p>
 +
            </td>
 +
            <td width="208" valign="top">
 +
                <p>
 +
                    Notebook
 +
                </p>
 +
            </td>
 +
        </tr>
 +
        <tr>
 +
            <td width="208" valign="top">
 +
                <p>
 +
                    Electroporation
 +
                </p>
 +
            </td>
 +
            <td width="208" valign="top">
 +
            </td>
 +
            <td width="208" valign="top">
 +
            </td>
 +
        </tr>
 +
    </tbody>
 +
</table>
 +
<p>
 +
    <strong><u>Theoretical background</u></strong>
 +
    :
 +
</p>
 +
<p>
 +
    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.
 +
</p>
 +
<p>
 +
    <strong><u>Procedure</u></strong>
 +
    <strong>:</strong>
 +
</p>
 +
<p>
 +
    1. The ingredients prepared according to the protocol with the GFP gene
 +
    plasmid:
 +
</p>
 +
<p>
 +
    23 ul of PUC GFP DNA ,187 ul OF 1M CaCl2, DDW up to 750ul + 750 HEBSX2
 +
</p>
 +
<p>
 +
    2. All ingredients were made and then filtered in 0.22 filter for sterile
 +
    solution
 +
</p>
 +
<p>
 +
    3. 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)
 +
</p>
 +
<p>
 +
    4. In the 6 wells there were 2 options for the transformation:
 +
</p>
 +
<p>
 +
    a) Medium removed, 250ul reagent added. after 30 min new medium(2.5ml)
 +
    added
 +
</p>
 +
<p>
 +
    b) 250ul reagent added + old medium(2.5ml)
 +
</p>
 +
<p>
 +
    5. After 8 hours all old medium removed and added new 2.5ml of relevant
 +
    medium
 +
</p>
 +
<p>
 +
    <u></u>
 +
</p>
 +
<p>
 +
    <strong><u>Design</u></strong>
 +
    <strong>:</strong>
 
</p>
 
</p>
 
</div>
 
</div>

Revision as of 11:34, 10 October 2018

OriginALS

OriginALS

Collaborations

HebrewU
Tec De
Trinity
US
MichiganState

Tech

HebrewU / HujiGEM

Calcium Phosphate Transfection

Title :
Transfection to microglia and astrocytes via “calcium phosphate” method

Conducted by :
Sagi

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 electrophoresis) in order to find an efficient way of inserting our plasmids into astrocytes and microglia for the continuation of the project.

Experiments

Protocols

Notebook

Electroporation

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 :

1. 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

2. All ingredients were made and then filtered in 0.22 filter for sterile solution

3. 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)

4. 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)

5. After 8 hours all old medium removed and added new 2.5ml of relevant medium

Design :

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.

OriginALS postcard as part of the collaboration with the Duesseldorf team.

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.

OriginALS

About Us


The BGU-iGEM team “OriginALS” hopes to develop an innovative therapeutic approach to prolong the life expectancy of ALS patients, using Synthetic Biology. We are dedicated to promoting ALS awareness and research in Israel through public engagement and educational activities.

    Keep in Touch

  • @iGEMBGU
  • @igem_2018_bgu
  • @originalsbgu
  • originalsbgu@gmail.com

    Address

  • Ben-Gurion University of the Negev
    Ben Gurion 1, Beer Sheva 8410501, Israel