Team:UC Chile/Cyano/Labook/august

August

During the last days of July we assembled and got PCR confirmation of BBa_K743006 (RS1-KanR-B0015-RS2). The construct was purified and now is part of our BioBrick collection. Parts C4 construct, psbAB (Synechocystis promoter) and RFP marker were assembled by Gibson and subsequently transformed. In this way we obtained BBa_K743004 (RS1-psbAB-mRFP-B0015-KanR-B0015-RS2). Confirmation was positive by PCR and digestion. Device (BBa_K743004) was transformed into cells and then purified. Later it was used to amplify its vector backbone from RS2 to mRFP. By doing this we wanted to exchange part KanR for KanRi (KanR inverse). The new amplicon was purified (VB to mRFP) and was assembled to KanRi part by Gibson, obtaining the device BBa_K743009 (RS1-psbAB-mRFP-B0015-KanRi-B0015-RS2). This new construct was transformed into cells and subsequently results were confirmed by PCR and digestion. In addition, we obtained circadian promoters pta, pcaa3 and psigE by PCR from Synechocystis DNA. LuxAB from Xenorhabdus luminescens was amplified. By assembly of the parts pta, LuxAB (from Xenorhabdus luminescens) and BBa_K743009 VB (amplified from KanRi to RS1) we obtained the device BBa_K743014 (RS1-pta- LuxAB -KanRi-B0015-RS2). Result was confirmed by digestion. Afterwards, we amplified BBa_K743014 (from KanRi to pta) in order to exchange LuxAB from Xenorhabdus luminescens for LuxAB from Vibrio fischeri. This was accomplished by Gibson assembly obtaining BBa_K743015. Result was confirmed by digestion. The parts circadian promoter pcaa3 and psb1C3 vector backbone were amplified and further assembled by Gibson. Subsequently, cells were transformed to obtain promoter pcaa3 in BioBrick format (part: BBa_K743002). Result was confirmed by digestion. The following constructs were confirmed by sequencing BBa_K743009 BBa_K743004 BBa_K743002 BBa_K743016 BBa_K743014

First Synechocystis transformations were attempted using Int_C BBa_K743004 BBa_K743014 BBa_K743016 psbAB+GFP+pPMQAK1