Team:UC Chile/Cyano/Labook/september

September

BBa_K743015 assembled during the last days of August was verified by sequencing.

Also during the last days of August, Synechocystis PCC 6803 was transformed with:

Int_C BBa_K743004 BBa_K743014 BBa_K743016 psbAB+GFP+pPMQAK1

Only Int_C could not be transformed into cells. A colony PCR was run to amplify different sequences of BBa_K743004 and psbAB+GFP+pPMQAK1. Positive results for only for para BBa_K743004.

BBa_K743014 was inoculated in a 500 mL flask to test induction by decanal. The inoculum grew in Kanamycin but did not produce bioluminescence when induced by decanal.

BBa_K743004 and psbAB+GFP+pPMQAK1 was replated in Kanamycin plates but there was no growth. Construct psbAB+GFP+pPMQAK1 was replated in Kanamycin three times, but never grew. We believe it could not be transformed.

We transformed Synechosystis pcc 6803 again with the following parts and now cells are being incubated at optimal conditions (continuous light and 30 °C).

BBa_K743004 BBa_K743009 BBa_K743016 BBa_K743006 BBa_K743014 BBa_K743015 IntC Control – (KAN) Control – (CLO)

IntC could not be transformed again. The rest of the transformations seem ok.

BBa_K743014 and BBa_K743016 were plated in kanamycin plates from transformation plates and a colony PCR was run for BBa_K743014 amplifying LuxAB and LuxAB with promoter.

A new round of Synechocystis was run under the same conditions and constructs that the last one.

The last two transformations resulted in negative controls and individual colonies for most constrcuts except for IntC (did not transform). Colonies were picked from all transformations and were replated in kanamycin plates. Our cells grew in kanamycin, which indicates they are transformed. Also, the plates were inoculated I nliquid media with kanamycin and they also grew.

After multiple replatings in Kanamycin, a massive colony PCR was run of the transformed cells from the last two rounds of transformation. BBa_K743015 and BBa_K743016 transformations were verified.

Due to the important amount of failed attempts to transform IntC plasmid from Utah, this was digested resulting in bands of wrong size. We sent it to sequencing.

Characterization of LuxBrick was done.

A growth curve was characterized for Synechosystis PCC 6803 from an OD730:0.4800 inoculum.

Inoculums: 3 flasks with 1 mL in 150 mL of normal BG11. 1 control with 1 mL in 150 mL of BG110.

• Shaker at 90 RPM.

Experiment for characterization of Gibson for small parts was run.

We tried to see LuxBrick induced under microscope. We concluded bioluminescence can’t be seen under microscope.

We amplified parts and run a Gibson to assemble BBa_K743017 and BBa_K743018. Transformation and verification by digestion and PCR was done afterwards. Constructs were sent to sequence.

Biolumiscence of different constructs with luciferase was measured when induced with different aldehides (decanal and dodecanal) using an Illuminometer.