Part:BBa_K082034:Experience

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Characterization of BBa_K082034 by ETH Zurich 2010 iGEM Team

Introduction

The iGEM 2010 team of ETH Zurich considered this part as a reporter and therefore made an effort to characterize it. We evaluated two plasmids for expression of the part, a high copy plasmid (pSB1A2) and a medium copy plasmid (pSEVA).

Plasmids

plasmid	origin	resistance	additional information
pSB1A2	high copy	amp
pSEVA132	approx. 75 copies/cell	kan	[http://2010.igem.org/Team:ETHZ_Basel/Biology/Implementation copy number]
pKQV4		tet, amp	[[1]]; contains lacIq gene with constitutive promoter

BBa_K082034 in pSB1A2

Methods

An initial culture of E. coli DH5α (5 ml LB in 15 ml Falcon tube) was incubated overnight on a shaker (37°C, 220rpm). From this initial culture 1 ml were transferred to 25 ml Falcon tubes containing 4 ml LB. After one hour of incubation induction was initiated by 5uM, 50uM, 500uM and 5 mM IPTG respectively. Fluorescence (excitation at 485nm and emission at 530nm) and optical density at 595 nm was measured after two hours of incubation with a PerkinElmer Victor3 Fluorometer.
From the measured fluorescence the fluorescence of an LB blank was substracted and then divided by the difference in optical density between the sample and the LB blank. The obtained values were normalized by the control (DH5α cells not carrying the plasmid).

Results

relative fluorescence of pSB1A2. The fluorescence of E. coli cells containing pSB1A2 compared to E. coli without plasmid. Inducer level did not affect fluorescence.

Conclusion

It seems that the endogenous level of LacI is not sufficient to repress the part efficiently. Thus, the fluorescence observed resulted from leaky expression, while the effect of the inducer was probably hidden behind noise. This plasmid seems suitable to evaluate the presence or absence of the plasmid.

BBa_K082034 in pSEVA132

Methods

In order to prevent leaky expression of the part the plasmid pKQV4 was introduced in addition to pSEVA132. pKQV4 contains a LacI repressor gene, which is constitutively expressed.
From an initial culture of E. coli DH5α cells (5 ml LB in 15 ml Falcon tube, incubation overnight at 37°C, 220rpm) cultures (10 ml LB in 100 ml Erlenmayer flask) were inoculated to an OD (at 600 nm, using an Eppendorf Biophotomer) of 0.05. After 1 hour of incubation (37°C, 220rpm) expression was initiated by 1mM IPTG.

Results

BBa_K082034 Cell density over time with pSEVA132.	BBa_K082034 Fluorescence over time with pSEVA132. Induction at 60 min.	BBa_K082034 Fluorescence per Cell density over time with pSEVA132. Induction at 60 min.
BBa_K082034 Cell density over time with pSEVA132. No induction.	BBa_K082034 Fluorescence over time with pSEVA132. No induction.	BBa_K082034 Fluorescence per Cell density over time with pSEVA132. No induction.
BBa_K082034 Cell density over time with pSEVA132 and pKQV4.	BBa_K082034 Fluorescence over time with pSEVA and pKQV4. Induction at 60 min.	BBa_K082034 Fluorescence per Cell density over time with pSEVA132 and pKQV4. induction at 60 min.

Conclusion

Cells containing only pSEVA132 and no pKQV4 showed some leaky expression when not induced. However, cells containing pSEVA132 and pKQV4 didn't show any expression even at the inducer concentration of 1 mM. The reason for this is probably the elevated endogenous level of LacI provoked by the additional pKQV4 plasmid.

Reference

[1] [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC400994/pdf/emboj00129-0314.pdf Strauch, M. A.; Spiegelman, G. B.; Perego, M.; Johnson, W. C.; Burbulys, D.; Hoch, J. A. The transition state transcription regulator abrB of Bacillus subtilis is a DNA binding protein. EMBO J. 1989, 8, 1615-1621.]

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