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 constitutively expressed reporter in order to verify the success of a special [http://2010.igem.org/Team:ETHZ_Basel/Biology/Cloning cloning strategy]. We therefore made an effort to characterize it. Since the part contains a lacI binding site, the capacity of endogenous LacI for repression was evaluated. Two plasmids for the expression of the part were analyzed, pSB1A2 (high copy plasmid) and pSEVA132 (medium copy).

Cloning

control digest of pSEVA132.

digest of pSB1A2.

Since the part BBa_K082034 was distributed in the plasmid pSB1A2 it could readily be used for the experiments and did not have to be cloned further. pSEVA132 required some preparation. First pSB1A2 and pSEVA132 were digested according to the protocol found [http://openwetware.org/wiki/Engineering_BioBrick_vectors_from_BioBrick_parts/Restriction_digest here]. The restriction enzymes used were EcoR1 and Pst1. The part was then isolated from pSB1A2 with an agarose gel and ligated into pSEVA according to the [http://www.neb.com/nebecomm/products/protocol2.asp quick ligation protocol] of New England Biolabs. Chemically competent E. coli DH5α cells were transformed by the [http://www.neb.com/nebecomm/products/protocol3.asp transformation protocol] of New England Biolabs.
control digest of pSB1A2. Lane 1:

Plasmids

plasmid	origin	resistance	additional information
pSB1A2	pMB1; 100-300 copies/cell	amp	link to registry
pSEVA132	pBBR1; approx. 75 copies/cell	kan	From Victor de Lorenzo's lab; to see the analysis of the [http://2010.igem.org/Team:ETHZ_Basel/Biology/Implementation#Experimental_realization copy number] visit the link (pSEVA132 = wv1)
pKQV4	pBR322	tet, amp	[1]; contains lacIq gene under a 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

Cell density over time with pSEVA132.	Fluorescence over time with pSEVA132. Induction at 60 min.	Fluorescence per Cell density over time with pSEVA132. Induction at 60 min.
Cell density over time with pSEVA132. No induction.	Fluorescence over time with pSEVA132. No induction.	Fluorescence per Cell density over time with pSEVA132. No induction.
Cell density over time with pSEVA132 and pKQV4.	Fluorescence over time with pSEVA and pKQV4. Induction at 60 min.	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 did not 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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