Document

Notebook

No activity in lab this week

Reunión with the team to determinate the list of protocols that we are going to get for the posterior lab work.

No activity in lab this week

It is possible to elaborate a first document with the experimental strategy and the most of the protocols. It is presented and evaluated in front of professor ÁLvaro, who suggested us correct some details and make contact Dr. Tomás egaña, Engineer in Biotechnology molecular , expert working with the microalgae Chlamydomonas reinhardtii.

Reunion with the team to find the answers of the doubts about the document with the elaborated protocols.

Reunion with Dr. Tomás Egaña, who, kindly, help us to solve our doubts about our experimental strategy and some protocols related with Chlamydomonas reinhardtii. Finally, he talk us about Myra Chávez, a postdoc and an expert in work in lab with Chlamydomonas reinhardtii, who can share with us transformation plasmid, and other materials.

We accomplished to have contact with Myra Chávez, who offered help solving some doubts and she accepted give us pBC1-CrGFP. From now, Myra became a big help and support for our team and we nicknamed her as Maymi.

No activity in lab this week

Reunion to work in the sequences that must be sent to print, define them, analysis them, and search the tools of optimization of codons for chlamydomonas reinhardtii to make the first order to IDT.

Improve of the genetic circuit. Maymi accept be part of the team.

Use the IDT tool to make the codon’s optimization.

Collaboration from Germany: LMU München (Ludwig Maximilians Universität München), they help us checking our experimental strategy and sequences design.

Reunion with Jose Duguet, who give us information about how to make the iGEM parts and help us with some doubts. Work in the parts design.

Reunion with professor Álvaro, design of the primers, and consultation

Collaboration from England, Dra Allison Smith from the Cambridge University. She shared us the promoter MetE sequence, 5’UTR and its terminator. Also, gave us some advices for the construction of our construct. Finished the primers design.

The sequences are finally ready to be sent to IDT.

Is made the shipment of the sequences to IDT.

Reunion with Maymi and professor Álvaro, to specify the experimental strategy, materials and required methods.

Amplification of pBC1-CrGFP in e. coli DH10B chemically competent.

Repetition of the last lab. Because there was not growth on the plate, was repeated the amplification, however, now the transformation is made in the e coli top 10 chemi competent strains and with more care and caution. After a thermic shock and the incubating in TB medium for an hour and a half, the cell were plated in several plates with LB and ampicillin, and they are left incubating at 37°C.

The plates are checked and if there is presence of transformants colonies with resistance to ampicillin, two of them are inoculated in Falcon tubes with Lb and ampicillin medium at 37°C.

Miniprep of the transformants colonies in Falcon. Isolation of the plasmid pBC1-CrGFP.

Test transformation of the chlamydomonas reinhardtii by the method of glass beads with pBC1-CrGFP. Also was learnt the cell counting on a Neubauer camera. After the transformation the cells were left incubating in Falcon tubes with TAPS medium in dark during the night at ambient temperature with agitation.

Continuing with the last lab. The cells were incubated in TAPS, then they were centrifuged and sowed in plates with TAPS medium with agar and paromomycin. They were left incubating at ambient temperature.

No activity in lab this week

Due to was informed that some of the sequences that were sent could not be printed, a second order was made to IDT with the correct sequences.

Arrived the first order from IDT.

Resuspension of the lyophilized gBlocks.

Was informed to us that one of the sequences from the second order can not be synthesized again.

Resuspension of the universal primers and PCR of the gBlock of the first order from IDT.

Preparation of gel extraction of amplified sequences cut DNA bands.

Extraction and purification of DNA from the agarose bands gels.

Digestion of the standard sequences and the igem vector pSB1C3. Ligation with the standard sequences.

Transformation by electroporation of the vectors and sequences in BL21DE3 E. coli strain. Sow in selective mediums, they were left incubating at 37°C until 2 p.m. of the next day.

Rescue of the colonies and growth in LB liquid medium with antibiotic. Left at 4°C

Miniprep of the colonies and generation of a glycerol inventory.

First test of miniprep digestion. The samples were saved at -20°C.

Preparation of visualization gel of the miniprep digestion. Was not observed bands on the gel.

Was repeated the visualization gel and again was not observed the bands on the gel. The part was sent.

Was decided to repeat the test of miniprep digestion and was made the electrophoresis. Finally, was observed the bands, which validated the construct with the part that is going to be sent.

No activity in lab this week

ono

Team:UChile OpenBio-CeBiB/Notebook

Notebook