Best Basic Part

For this year’s iGEM competition, our team has chosen to present Calcineurin-dependent response element (CDRE) (BBa_K2207021)and Mature serine protein from Paecilomyces lilacinus (BBa_K2207023) for the award of the basic part.

CDRE

CDRE，calcineurin-dependent response element，is a segment of DNA sequence which can be regulated by specific transcription factor. This TF is activated by calcineurin in the present of calcium ion. To create a promoter can be up-regulated by calcium influx，we replaced the upstream activating sequence(UAS) of CYC1 promoter with 4 CDREs. This promoter is designed for Saccharomyces cerevisiae and it depends on S.cerevisiae's endogenous calcineurin and calmodulin^[1][2]. We choose mRFP as the report gene. We cultured the yeast in calcium-inducing medium and uninduced medium which contains a relatively low concentration of calcium ion (1xYPD medium).

Obviously, the transgenic yeast cultured in the calcium-inducing medium(200mM Ca²⁺) is turning red while the control shows no significant changes. Then we detected the fluorescence of this two cultures. To eliminate the influence of the concentration of the yeast, we calculated the value of Fluorescent Intensity/OD600 to evaluate these two groups.

The value indicated that the fluorescence intensity of the calcium-induced group has improved about 147%. It’s not quite a huge change and the uninduced group has already shown a great intensity of fluorescence. Then we detected the intracellular calcium ion concentration with Fluo 4-AM whose fluorescent intensity can represent the relative concentration of calcium ion.

Fig.1 (a) Relative fluorescent intensity of two groups. (b) Relative calcium content in two groups

As the result shows, 8% change of the intracellular ion concentration can contribute to 147% higher expression level of the downstream gene. It seems that our CDRE promoter is much more sensitive than we expected.

Mature serine protein

Root-knot nematodes (Meloidogyne spp.), which are one of the most destructive nematodes, cause the loss of crop about 10%, serious as high as 75%. Like the insect cuticlehe, nematode eggshell consists mainly of proteins and chitins. The egg-parasitic fungus P.lilacinum secretes protease and chitinase to hydrolyze the nematode eggshell, so that the root knot nematodes cannot grow normally^[3]. Among them, serine protases plays an important role in hydrolyzing the eggshell of root-knot nematodes.

The gene which can express serine protease in yeast was synthesized by Genscript. Before synthesizing this gene, we did codon optimization based on the codon preference of yeast and added a flag-tag to the N-terminal of the serine protease.After extracting the whole proteins of the yeast which transferred plasmid successfully, we performed western-blot and checked the serine protein was expressed in the yeast. (Result is as follow) The band was very shallow, in other words, the concentration of the serine protease was very low.

Fig.2 Western-blot result

The band with red circle was the band of the serine protease and the marker was a protein marker of aidlab.

In order to test whether the serine protease could work normally in the yeast, we performed the enzyme activity detection using BAEE solution. We did two sets of experiments: one added PMSF, which was a inhibitor of serine protease, and the other did not. And then, reading the OD253 of these two solutions.(You can know more details about the detection from the protocol) Obviously, the OD253 of the former one is higher than the later one and the values of OD253 increased with time in a period of time ; therefore, we made the conclusion that the yeast produced the serine protase successfully and effectively.

Fig.3 The values of OD253 increased with time

The values of OD253 increased with time and this solution did not add the PMSF.

Fig.4 A box-plot of the OD253

These were the OD253 of the solutions after a period of reaction.The red one was the solution that did not add the PMSF; the blue one was the solution that added. Obviously, the OD253 of the former one is higher than the later one, so that, we could say that the yeast produced the serine protase successfully and effectively.

This year we submitted several well-working basic parts to the BioBrick Registry.

Table of the Basic Parts we submitted to the BioBrick Registry

Name	Type	Description	Designer	Length(bp)	Submitted
BBa_K2207003	Composite Parts	Trichoderma HR System I SOD-Tubulin dobule promoter	Yihe Zhang	3155	✔
BBa_K2207004	Composite Parts	Trichoderma HR System II Ech42-H3 double promoter	Yihe Zhang	4845	✔
BBa_K2207005	Composite Parts	Trichoderma HR System III L1-ADH1-RP27-L2	Yihe Zhang	903	✔
BBa_K2207006	Composite Parts	Trichoderma HR System IV L3-ADH1-RP27-L4	Yihe Zhang	903	✔
BBa_K2207007	Composite Parts	Trichoderma HR System V Homologous Binding SiteA	Yihe Zhang	1676	✔
BBa_K2207008	Composite Parts	Trichoderma HR System VI Homologous Binding SiteB	Yihe Zhang	1650	✔
BBa_K2207009	Coding Sequences	PhlF transcription factor	Qianjin Jiang	627	✔
BBa_K2207010	Other	Phl Operator	Yuxing Chen	825	✔
BBa_K2207021	Promoters	Calcineurin-dependent response element (CDRE)	Shisheng Li	474	✔
BBa_K2207023	Coding Sequences	Mature serine protein from Paecilomyces lilacinus	Zifan Xie	882	✔
BBa_K2207024	Promoters	T7-mutant1	Yuxing Chen	42	✔
BBa_K2207025	Promoters	T7-mutant2	Yuxing Chen	42	✔
BBa_K2207026	Promoters	T7-mutant3	Yuxing Chen	42	✔
BBa_K2207027	Promoters	T7-mutant4	Yuxing Chen	42	✔
BBa_K2207028	Promoters	T7-mutant5	Yuxing Chen	42	✔
BBa_K2207029	Promoters	T7-mutant6	Yuxing Chen	42	✔
BBa_K2207030	Promoters	T7-mutant7	Yuxing Chen	42	✔
BBa_K2207031	Coding Sequences	PhlE efflux pump	Junming Qian	1272	✔

Table.OD600 reference point

Reference

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[1] Cyert M S. Calcineurin signaling in Saccharomyces cerevisiae: how yeast go crazy in response to stress[J]. Biochemical and biophysical research communications, 2003, 311(4): 1143-1150.

[2] Cyert M S. Genetic analysis of calmodulin and its targets in Saccharomyces cerevisiae[J]. Annual review of genetics, 2001, 35(1): 647-672.

[3] Brand D, Roussos S, Pandey A, et al. Development of a bionematicide with Paecilomyces lilacinus to control Meloidogyne incognita.[J]. Applied Biochemistry & Biotechnology, 2004, 118(1-3):81-88.