Team:Tokyo-NoKoGen/Project/aggregation
From 2010.igem.org
Introduction
Why is this device needed?
- Taking up a target material in E. coli.
- Self-aggregation of E. coli. : Skipping the harvest work.
What is this device composed inside it?
How does this device work in EcoTanker?
This device will be activated by the Green light receptor (detail is on the page of PHOTOCONTROL). When we want to collect E. coli, we will flash green light and aggregate it (Fig. 3).Progress
The wild type of Antigen 43 contains 6 PstI sites. So, to use as a BioBrick, we had to modify this 6 PstI sites. To modify, we choose Overlap PCR and the scheme will be below. First, we amplify 3 fragments by using the genome as a template and primers to modify the PstI sites. Then, by using these 3 fragments as a primer, and the forward and reverse primer when we cloned Antigen 43, we amplified 4 fragments. And we performed overlap PCR with this 4 fragments by using the forward and reverse primer. We confirmed the amplification of the gene of Antigen 43. After digesting this fragment with EcoRI and PstI, ligated to pSB1C3 to construct the plasmid pSB1C3-Antigen 43(BBa_K317008). We analysis the sequence and confirmed that Antigen 43 didn’t have any PstI sites. By using this new BioBrick, we constructed transcriptional unit, generator, and 4 devices. We also submitted these 6 parts as a new BioBrick(Table).
We confirmed the function of Antigen 43 by using 2 devices that expresses Antigen 43 constitutively and evaluate by measuring OD660 approximately 1 cm from the top [1]. The methods will be, inoculate a singe colony to 5 ml of LB medium and incubate at 37℃ over night. Then, stopped incubation and stand the culture solution. Then, measured the OD660 from the top of 1 cm of the culture. The results of measuring OD660 (Fig. 5) and a picture of appearance of E. coli sinking will be shown below (Fig.5). From the results, the E. coli that harbors the plasmids (Constitutive promoter-RBS-Antigen 43-Double terminator) show decreasing of OD660 although the strength of the promoter is weak (BBa_J23102 is strong and BBa_J23109 is weak). So, we can say low expression of Antigen 43 will be enough to sink the E. coli.
BioBricks we submitted are bellows.
References
[1] KRISTIAN KJÆRGAARD, MARK A. SCHEMBRI, HENRIK HASMAN, AND PER KLEMM. Antigen 43 from Escherichia coli Induces Inter- and Intraspecies Cell Aggregation and Changes in Colony Morphology of Pseudomonas fluorescens JOURNAL OF BACTERIOLOGY, 2000, p. 4789–4796, Vol. 182, No. 17