Difference between revisions of "Team:Linkoping Sweden/Notebook"

The Nordic iGEM Conference, NiC, is a weekend long mini Jamboree held by one of the Nordic iGEM Teams. This event is hosted by the team who held the best presentation the previous year according to the NiC jury. The 2016’s conference was arranged by Stockholm, the capital of Sweden. This was a perfect opportunity to get to know the Nordic iGEM Teams and to practice our presentation for the Giant Jamboree. Also, the event offered Workshops, BBQs with biotech students from five different countries amongst other activities.

In the waiting for ordered primers, vector and such, we prepared TAP and LB medium, agar plates etc.

We made the cover of the local newspaper, Östgöta correspondenten called Corren. This Newspaper is delivered to households each morning within the providence Östergötland. The article was mainly about the concept of iGEM and the project of the year. The focus laid at making more biomass out of C. reinhardtii and the contributing work to minimize the greenhouse effect.

With the laboratory deadline getting closer, we were still out of luck when it came to results. We decided that it was time to make a change regarding how we executed our project to reach our goals. The project idea remained the same, but the approach changed. Instead of using the traditional digest-ligation method to construct the parts, we went ahead with a different method called Gibson Assembly. This Assembly enables the creation of a construction of up to six parts in just one reaction. Our hope was restored!

We used the iGEM Competent Cell Test Kit in order to control the transformation efficacy of our heat competent cells (E. Coli Xl1-blue). Our goal was to introduce the DNA construct into the algae C. reinhardtii, which is why the first cultivation was started early. We wanted to optimize the cultivation conditions for the algae.

After inactivation of the gene coding for starch in a baby Mona our team mascot evolved into Miss Mona. This young single-celled lady alga is a specie called C. reinhardtii, the algae we use in our project experiments. Her mainly diet is Swedish bulk confectionary and she is currently single. Ms Mona was created both as a tradition for the Liu iGEM team, as well as a part of the Human Practice. She has been attending to all exhibitions and presentations of the year.

The first PCR-reaction was made and we amplified one of the desired sequences to our construct, the Cas9 sequence, from an existing BioBrick. Verification of the product was made according to fragment size through gel electrophoresis.

As the DNA construct was designed with the light inducible promotor, a test culture in darkness was grown in order to study the optimal parameters such as temperature, TAP medium etc. in these conditions.

Sweden's Television, SVT, is the national public TV broadcaster of Sweden. They made a reportage about our project in particular, but we also informed about iGEM in general.

We made the first attempt to assemble our final DNA construct with the method Gibson Assembly, which was totally new for us.

Gibson assembly is an exonuclease-based method to assembly multiple DNA fragments in correct order. The method was invented in 2009 by Daniel G. Gibson, of the J. Craig Venter Institute. The assembly reaction is carried out in one single reaction-tube, all at once, at 50° Celsius for 15-60 minutes. The process involves three different enzymatic actions. A 5’ exonuclease creates overhangs, enabling matched fragments to anneal. Then a DNA polymerase fills gap between the annealed strands and the 5´ end. Finally, a DNA ligase seals the gaps between the filled in gap and the annealed strands.

For more information, see Gibson Assembly

Using this method, we constructed three different parts, see results.

After the second Gibson Assembly we succeeded to bring together a variant of our final DNA construct. This allowed us to try out the electroporation of this fragment into the algae C. reinhardtii. Confocal microscopy was used to detect fragment-positive colonies for yellow fluorescent protein (YFP), while the YFP was inserted to the final DNA construct instead of the Hygromycin B cassette.

We held a presentation for a course in gene technology at the University. The presentation was a preview of the presentation that we are about to have on the Giant Jamboree. We also promoted iGEM and the yearly LiU iGEM project, many found a great interest in taking part in this synthetic biology project and signed up for a future membership.

The major part of our team attended to the study visit at Ångströmhuset, where the university have their electron microscope. The building is named after the Swedish physicist Anders Ångström and is located a short distance from the centre of the university. The location as well as the placement on solid ground is to minimize disruption of the measurements. We learned a lot about the function and the applications of electron microscopy, but also how difficult it can be to receive trust-worthy data from measurements we use daily.