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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 cytosolic LacI for repression was evaluated. With the aim to outcompete cytosolic LacI two plasmids with elevated copy number for the expression of the part were analyzed: pSB1A2 (high copy plasmid) and pSEVA132 (medium copy).

Cloning

digest of pSB1A2.

control digest of pSEVA132.

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 EcoRI and PstI. The part was then isolated from pSB1A2 with an agarose gel and ligated into pSEVA132 according to the [http://www.neb.com/nebecomm/products/protocol2.asp quick ligation protocol] of New England Biolabs to give rise to the plasmid pSEVA132_BBa_K082034. 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: [http://www.neb.com/nebecomm/products/productn0468.asp 1kb ladder]; lane 2: digested pSB1A2, part at 1.1kb, vector at 2kb.
control digest of pSEVA213. lane 1 and 6: [http://www.neb.com/nebecomm/products/productn0468.asp 1kb ladder]; lane 2 and 4: pSEVA132_BBa_K082034 not digested; lane 3 and 5: digested pSEVA132_BBa_K082034, part at 1.1kb, vector at 4.5kb.

Plasmids

BBa_K082034 in pSB1A2

Methods

Relative fluorescence of pSB1A2. The fluorescence of E. coli cells harboring pSB1A2_BBa_K082034 compared to E. coli cells without plasmid after 120min of incubation at an IPTG concentration of 5mM.

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 Isopropyl-β-D-thiogalactopyranosid (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

E. coli DH5α cells harboring pSB1A2_BBa_K082034 showed an increase of fluorescence by a factor of around 6 compared to E. coli DH5α cells not containing the plasmid (see picture on the right). As a representative the culture with an induction level of 5mM is shown. However, inducer concentration did not have an effect on 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. pSB1A2_BBa_K082034 seems suitable for constitutive expression of GFP. The experiment would need to be repeated in order to get significant results.

BBa_K082034 in pSEVA132

Methods

In order to prevent leaky expression of the part the plasmid pKQV4 was introduced in addition to pSEVA132_BBa_K082034. 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 of E. coli DH5α harboring pSEVA132_BBa_K082034 over time.	Fluorescence of E. coli DH5α harboring pSEVA132_BBa_K082034 over time. Induction at 60 min.	Fluorescence per cell density over time of E. coli DH5α harboring pSEVA132_BBa_K082034. Induction at 60 min.
Cell density of E. coli DH5α harboring pSEVA132_BBa_K082034 over time. No induction.	Fluorescence of E. coli DH5α harboring pSEVA132_BBa_K082034 over time. No induction.	Fluorescence per cell density over time of E. coli DH5α harboring pSEVA132_BBa_K082034. No induction.
Cell density of E. coli DH5α harboring pSEVA132_BBa_K082034 and pKQV4_lacIq over time.	Fluorescence of E. coli DH5α harboring pSEVA132_BBa_K082034 and pKQV4_lacIq over time. Induction at 60 min.	Fluorescence per cell density over time of E. coli DH5α harboring pSEVA132_BBa_K082034 and pKQV4_lacIq. induction at 60 min.

Conclusion

Cells harboring only pSEVA132_BBa_K082034 and no pKQV4_lacIq showed some leaky expression when not induced. However, cells containing pSEVA132_BBa_K082034 and pKQV4_lacIq did not show any expression even at an inducer concentration of 1 mM. The reason for this might be the elevated cytosolic level of LacI provoked by the additional pKQV4_lacIq plasmid.
For constitutive expression of GFP the system with pSEVA132_BBa_K082034 could be useful.

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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