Team:Tartu TUIT/Notebook

Searched for the pathway of synthesis of mycosporine-like amino acids from the genes of different organisms. Actinosynnema mirum DSM with the gene names amir_4256, amir_4257, amir_4258, amir_4259. And Nostoc commune with the gene names mysA, mysB mysC and mysD.

iGEM distribution kit arrived.

Ordered synthetic DNA to initiate the lab work process.

Ordered primers for biobricks.

Primers arrived.

Started the first cloning process Biobricks with PCR, followed by restriction and overnight ligation.

First bacterial transformation. Resulting colonies on most of the plates.

Obtained mysA biobrick sent for sequence determination. Positive result.

Interlab: we took two colonies from each of transofrmation plates and inoculated them in seperate tubes of 5 mL of LB+CAM.

Team photoshoot.

CPEC, gybson, vegas cloning initiation. PCR with Phusion Polymerase.

H2O 31.6
5xHF buffer 10
25mM dNTP's (,,Template DNA) 0.4
Forward primer 2.5
Reverse primer 2.5
Template DNA 1
DM50 1.5
Phusion polymerase 0.5

-Skype conference with Ecuador team

Successfully completed interlab. �Achievement- Fulfilled the bronze metal requirement.

Primers for gene one by one insertion, After having realized that instead of constructing a complex of wished genes and insert it to another does notlead to anything, We started to approach our goal by changing our mechanism and switching to one by one insertion of genes.

Started using the blue-white screening technique while transforming samples to the plates.

Lab technician day.Prepared LB media, different volume tips for autoclaving.

No colonies present of p8, p9, p10. Restared with digestion of those 3 for 3 days with ECORi PSTI restrictases.

p37, p39, p41. LIac mediated yeast transformation. First transformation into the yeast. Successful.

last transformation to the yeast.

Characterization of the biobrick BBa_K2576009 (pRPL18B promoter).

Strains used:

  • YST1 (CEN.PK-2-1C: MATa ura3-52 trp1-289 leu2-3,112 his3 [pRS306, ApaI])
  • YST2 (CEN.PK-2-1C: MATa ura3-52 trp1-289 leu2-3,112 his3 [pRS306 pRPL18B-EGFP-tCYC1,ApaI])
  • YST3 (CEN.PK-2-1C: MATa ura3-52 trp1-289 leu2-3,112 his3 [pRS306 pTDH3-EGFP-tCYC1,ApaI])