Study on Regulation of lux Genes by HapR from Vibrio orientalis
| Autor: | Tiara Eka Ariestantia, 堤心亞 |
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| Rok vydání: | 2017 |
| Druh dokumentu: | 學位論文 ; thesis |
| Popis: | 106 The process of communication from one cell to another in bacteria called Quorum Sensing (QS). This process can regulate the genes that must be perform in a group of bacteria. In V. orientalis, the luminous bacteria from Yellow coast of China there are six genes that linked with QS. That genes were: luxP (VIA_001151), luxQ (VIA_001152), luxS (VIA_003963), luxO (VIA_003573) and (VIA_003054), luxU (VIA_003574) and hapR (VIA_000273). This research focused on hapR gene since it was LuxR homologues and previous report showed that LuxR is an activator of the lux operon in V. harveyi (Swartzman et al., 1992; Showalter et al.,1990). LuxR in V. harveyi is not homolog with the luxR in A. fischeri, since it was the QS master regulator that controls expression of the genes in the QS of V. harveyi. This present study observed the role of HapR in bioluminescence process of V. orientalis. For confirmation, the interaction between HapR protein and promoter lux operon, the in vitro, in vivo and in silico study were done. DNA size of hapR was 615 bp and the protein size was about 24 kDa in pET28a, and around 19 kDa in pET29a. But only the pET28a that harbouring HapR that was protein fusion. The best treatment of IPTG induction for HapR was 1 mM IPTG in 25oC for 3 hour, and the buffer for purification the protein was Tris-HCl pH 8. In vitro study proved that HapR has an interaction with the promoter lux operon no.1-4 but not with the promoter lux operon no.5 and 6. It means upstream of the promoter lux operon no. 5 (292 bp) are sufficient to bind with HapR protein. In vivo study result showed that clones that has the higest Relative Light Unit (RLUs) was in HapR-PM with IPTG induction group, which is HapR-PM2 with 24.563 x 106 RLUs at 5 hour after IPTG induction. This result suggest that HapR bind the promoter lux operon around 792 bp-581 bp. In silico study result suggest that V. orientalis CIP 102891 = ATCC 33934 strain CIP 102891 has seven lux operon genes, luxCDABEGH and the arrangement is similar to V. harveyi strain ATCC 33843 (392 [MAV]) with 97.8% similarities. In silico study also demonstrated that V. orientalis only use one type of QS system that was AI2 system to manage their light production and it predicted that the regulation of bioluminescence was depend on phosphorylation-dephosphorylation cascade similar with AI2 system in V. harveyi and A. fischeri. So, it’s can conclude that HapR has a role on the regulation of lux genes from V. orientalis. |
| Databáze: | Networked Digital Library of Theses & Dissertations |
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