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Part:BBa_K082034:Experience

Designed by: Yanting Xue ,He Shen   Group: iGEM08_USTC   (2008-10-27)
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Characterization of BBa_K082034 by ETH Zurich 2010 iGEM Team

Introduction

The iGEM 2010 Team of ETH Zurich considered using this part as a 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).

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







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_BBa_K082034 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 BBa_K082034 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.

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 pSEVA132. 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.





pSB1A2_BBa_K082034

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 were 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 (E. coli 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). Inducer concentration did not have an influence on fluorescence which would be distinguishable from noise. As a representative the culture of E. coli DH5α harboring pSB1A2_BBa_K082034 induced at 5mM is shown.

Conclusion

It seems that the cytosolic level of LacI arising from chromosomally encoded lacI is not sufficient to repress the high copy plasmid pSB1A2_BBa_K082034. 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 challenge reproducibility and to obtain significant results.

pSEVA132_BBa_K082034

Methods

From an initial culture of E. coli DH5α cells (5 ml LB in 15 ml Falcon tube, incubation overnight at 37°C, 220rpm) containing either only pSEVA132_BBa_K082034 or pSEVA132_BBa_K082034 and pKQV4_lacIq, cultures (10 ml LB in 100 ml Erlenmayer flask) were inoculated to an OD (at 600 nm, measured with Eppendorf Biophotometer) of 0.05. Fluorescence (excitation at 485nm and emission at 530nm) and optical density at 595 nm were measured with a PerkinElmer Victor3 Fluorometer at time intervals of 15min. After 1 hour of incubation (37°C, 220rpm) expression was initiated by 1mM IPTG. The obtained values for fluorescence and optical density were corrected by the values of an LB blank.

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

In contrast to pSB1A2_BBa_K082034, cells harboring pSEVA132_BBa_K082034 seem to produce enough LacI in order to repress the load of BBa_K082034 brought to them, at least to some extent. Some leaky expression could still be observed. 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.
Our goal to use BBa_K082034 as a reporter for presence or absence looks quite easy. Either a high copy plasmid like pSB1A2 or a medium copy plasmid like pSEVA132 accompanied by IPTG would work. However, if a tightly regulated expression of BBa_K082034 is required levels of BBa_K082034 and of LacI would need to be carefully adjusted. Too much BBa_K082034 leads to leaky expression, as seen with the high copy plasmid pSB1A2_BBa_K082034 and also with the medium copy plasmid pSEVA132_BBa_K082034. Too much LacI, on the other hand, might completely block expression even if induced, as seen with pSEVA132_BBa_K082034 in combination with pKQV4_lacIq.
We could observe a delay between induction and the fluorescence gain of approximately 120min. The reason might be the time needed for correct folding of the GFP.

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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