Team:Tokyo Tech/Basic Part

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Basic Parts

Best Basic part amiE: BBa_K1949050

BBa_K1949050 meets the Silver Medal criteria!

amiE codes for protein AmiE. AmiE is an acylase that degrades long chain [N]-acyl homoserine lactone (AHL) molecules with acyl chains longer than six carbons. Prince coli(Fig. 4-2-1) expresses amiE, and Snow White coli recovers from its sleep and wakes up again. We tested the function of AmiE protein that influences the end of the story.

Fig. 4-2-1 AmiE degrades long chain [N]-acyl homoserine lactone (AHL) molecules
with acyl chains longer than six carbons

Our objective is to characterize the function of AmiE protein. We prepared three samples shown below. When we tested the AmiE degradation ability with these samples, the results show that C4HSL(refer to it as C4 hereafter) is not degraded by AmiE, but 3OC12HSL(C12) is degraded by AmiE(Fig. 4-2-2).

• PBAD - rbs - amiE (pSB6A1)

• Ptet - rbs - luxR - tt - Plux - rbs - gfp (pSB6A1)

• Ptet - rbs - rhlR - tt - Prhl - rbs - gfp (pSB6A1)




Fig. 4-2-2 AmiE degrades C12 but barely degrades C4

For more information, read this page !

Tokyo Tech 2016 iGEM Team: The Others Basic Parts

Name	Type	Description	Design	Length(bp)
BBa_K1949000	Regulatory	Pcold	Yoshio Takata	313
BBa_K1949020	Coding	yafN	Kazuki Fujisawa	297
BBa_K1949030	Coding	yafO	Yoshio Takata	402
BBa_K1949031	Transrational unit	rbs-yafO	Yoshio Takata	420
BBa_K1949050	Coding	amiE	Yoshio Takata	1476
BBa_K1949051	Transrational unit	rbs-amiE	Yoshio Takata	1494

Cold inducible promoter (Pcold): BBa_K1949000

BBa_K1949000 meets the Bronze Medal criteria!

This promoter is used to produce proteins effectively at low temperatures. This new promoter, a cold inducible promoter (we call this Pcold) consists of the cspA promoter, Cold Box, 5’-UTR, RBS and DB. The combination (of cspA promoter which is active at both low and high temperature, Cold box which inhibits excessive gene expression, 5’UTR which is stable at only low temperature, and DB which function as an extra RBS) activates gene expression at low temperatures.

RFU (Relative Fluorescence Units) of GFP / turbidity was measured by using cells cultured at 18℃ and 37℃ to confirm function of Pcold. The cells carry a plasmid which carries Pcold - gfp or Ptet - rbs - gfp.

Fig. 4-2-3 Pcold is a cold inducible promoter

E. coli cells which carry the Ptet - rbs - gfp plasmid was cultured at 18°C, and RFU of GFP was measured at indicated time points. Also, the same experiment was performed at 37°C. We thought this result was obtained because GFP is easily folded into correct structures at low temperatures. By contrast, RFU of E. coli which carried Pcold - gfp at 18°C was about eight fold higher than that at 37°C.(Fig. 4-2-3) From this result, we confirmed Pcold activates gene expression at low temperatures.

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yafN, yafO: BBa_K1949020, BBa_K1949030

BBa_K1949030 meets the Silver Medal crireria!

YafN is an antitoxin corresponding to toxin YafO, and YafN reverses inhibition of cell growth. yafN and yafO exist as an operon, and YafN also functions as a repressor in the operon. Therefore, yafNO operon’s translation is autoregulated by antitoxin YafN. When Lon protease senses environmental stresses such as DNA damage, the operon becomes active because of degradation of YafN by Lon protease.

Confirming YafO Function as Toxin on Agar Medium

We first confirmed YafO function by observing formation of colonies on agar medium. This experiment was carried out using E. coli which inducible yafO expression by arabinose and E. coli without yafO gene. Construction of plasmids used in this experiment is shown in Fig. 4-2-4 . We inoculated four types of E. coli differently on agar plates with and without arabinose. As a result(Fig. 4-2-4), all types of E. coli were able to form colonies on agar medium with no arabinose, and colonies of E. coli (c) and (d) had fluorescence of GFP. On the other hand, E. coli (a) and (c) couldn’t form any colonies on agar plates containing arabinose. From these results, we confirmed that all types of E. coli below functions as we expected.

Fig. 4-2-4 Confirming YafO function as toxin on agar plates

Toxin-Antitoxin Assay

From previous experiment, we were able to confirm that YafO works as toxin. Next, we confirmed whether the cell growth reverses by antitoxin YafN after inhibition by YafO. Construction of plasmids used in this experiment is shown in below. We prepared E. coli which can induce expression of yafO by arabinose and yafN by lactose. As comparisons, we also carried out same experiment with E. coli containing no yafO gene, no yafN gene and none of them. As a result, we couldn’t observe cell growth reversing after adding IPTG to induce expression of yafN, and their turbidity became constant as E. coli (c) containing no yafN. In this experiment, YafN couldn’t reverse the cell growth after inhibition by YafO. It seems to be difficult to control cell growth just like mazEF system, which demonstrated reverse of cell growth by antitoxin after function of toxin.

Fig. 4-2-5 toxin-antitoxin assay

For more information, read BBa_K1949020, BBa_K1949030 !