23.06–27.06

We first installed ourselves in the Pr. Zeeman lab and started by making some TAP-medium from C.reinhardtii following our protocol.

The first algal cultures were started and transferred to the LSL lab where we also made additional TAP-medium.

30.06–04.07

While waiting for our cassette fragments we made some additional algal cultures and started setting up the controlled temperature, light, and humidity room we had at our disposition.

We further investigated the optimal growth conditions of C.reinhardtii in this room.

07.07–11.07

The DNA material arrived on Monday and was first thawed and dissolved in nuclease free water.

Because the concentration measured by nanodrop was too low, we concentrated the fragment solution using a centrifugal evaporator.

The prepared samples were used to start assembling our cassette via Golden Gate assembly from the different fragments we designed.

The DNA of our Golden Gate assembly was used for transformation of E.coli.

14.07–18.07

The transformation failed, and the dry lab started designing a corrected version of the cassette.

A large amount of new cultures were started in the LSL lab to prepare for the autolysin extraction experiment.

Some first liquid culture media cultures were started and the autolysin extraction was attempted but failed due to a technical failure of the centrifuge.

An attempt to recycle used solid media algal culture was started.

21.07–25.07

We received the primers used for adding an overhang on our HDR arms in the cassette, as well as a backbone pTU1-A-RFD to replace our own designed backbone.

The Gibson assembly started by adding the overhangs to the digested fragment that contains the HDR arms. The product of that PCR was worked up by gel electrophoresis and was then further processed by gel extraction.

The extracted new HDR arms were used for a new Golden Gate assembly with our backbone and a new transformation was attempted.

The control with only the backbone was positive and some transformed bacteria were also present in the Golden Gate product transformation.

In the LSL the media recycling attempt failed and only gave very sparsely populated algae colonies. Therefore new plates were prepared for a future autolysin extraction.

28.07–01.08

A single colony of the transformed bacteria was prepared by miniprep and sent to sequencing for testing.

In the LSL a new autolysin extraction was performed and the sample was frozen.

04.08–08.08

Following the negative results of the new transformation plasmid, the HDR arms with overlap were sent to sequencing to test whether the Gibson assembly was successful.

In the LSL the activity of the extracted autolysin sample was investigated by testing its effect on healthy alga cell wall. The test was negative.

11.08–15.08

No major updates recorded.

18.08–22.08

Following the negative results of the HDR arms sequencing, our fragments were all sent to sequencing to test our starting material.

The results did not seem to show any problems with the starting material.

25.08–29.08

No major updates recorded.

01.09–05.09

No major updates recorded.

08.09–12.09

No major updates recorded.

15.09–19.09

In the LSL lab the first tests with culture media only containing CO2 as carbon source were conducted with different setups.

The trials covered automated syringe pressing, a balloon setup, and then a controlled balloon flow with clamping.

22.09–26.09

The first test with phosphate deprivation to test for REE salt binding was prepared. The REE salts were dissolved and a culture media was formed with them for treatment after the phosphate rich media.

29.09–03.10

The algae did not survive the three days in the phosphate poor buffer, so further analysis could not be done.

We repeated the transformation processes on E.coli with the new design of our cassette. Miniprep of the successfully transformed bacteria yielded a solution of our plasmid with a concentration of 198 ng/ul which was sent in for sequencing.

06.10–10.10

No major updates recorded.