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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 system to evaluate the [http://2010.igem.org/Team:ETHZ_Basel/Biology/Cloning cloning strategy]. Since the part contains a LacI binding site on the operator the expression behaviour under different concentrations of LacI and binding sites were examined.

Plasmids

plasmid purpose origin resistance additional information
pSEVA132 expression of BBa_K082034 pBBR1; approx. 75 copies/cell kan Victor de Lorenzo's lab; analysis of copy number (pSEVA132 = wv1)
pKQV4 expression of lacI pBR322; high copy tet, amp [1]







Cloning

digest of pSB1A2.
control digest of pSEVA132.

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.






Expression Behaviour of BBa_K082034 in pSEVA132

Methods

From an initial culture of E. coli DH5α cells (5 ml LB in 15 ml Falcon tube, incubation overnight at 37°C, 220 rpm) 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, path length 1 cm) of 0.05. Fluorescence (excitation at 485 nm and emission at 530 nm) and optical density at 595 nm were measured in a microtiterplate contatining 200 μl of samplte with a PerkinElmer Victor3 Fluorometer at time intervals of 15 min. After 1 hour of incubation (37°C, 220 rpm) expression was initiated by 1 mM IPTG. The obtained values for fluorescence and optical density were corrected by the values of an LB blank.

Results
pSEVA132_BBa_K082034, induction at 60 min.
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.
pSEVA132_BBa_K082034, no induction.
Cell density of E. coli DH5α harboring pSEVA132_BBa_K082034 over time.
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.
pSEVA132_BBa_K082034 and pKQV4_lacIq, induction at 60 min.
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 pSEVA132_BBa_K082034 showed an increase in fluorescence when induced with IPTG. However, if not induced, some leaky expression of BBa_K082034 could still be observed. In contrary, cells harboring pSEVA132_BBa_K082034 and pKQV4_lacIq did not show any increase in fluorescence, even at an inducer concentration of 1 mM. The increased level of LacI provoked by pKQV4 seem to shut off GFP production completely. 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.
If introduced into a medium to high copy plasmid, BBa_K082034 may only be useful to determine its presence/absence. 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 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.


Dependence of Expression of BBa_K082034 on Inducer Concentration

Methods

In order to reduce background fluorescence resulting from LB medium, M9 supplemented minimal medium enriched with thiamine and casamino acids was used in these experiments. It was prepared according to the Knight lab protocol.
All media used for growth of E. coli DH5α harboring pSEVA132_BBa_K082034 were complemented with 50 mg/ml of kanamycin. Incubation of cultures were carried out at 37°C on a shaking device (220 rpm).
For induction of the lacI operator Isopropyl β-D-1-thiogalactopyranoside (IPTG) was used. 3 different inducer concentrations were considered (10 μM, 100 μM and 1 mM of IPTG). Additionally, the fluorescence without any inducer was measured. As negative control served E. coli DH5α not harboring pSEVA132_BBa_K082034 induced by 1 mM IPTG. Measurements were made in triplicates.
Two starter cultures, one of E. coli DH5α harboring pSEVA132_BBa_K082034 and one of E. coli DH5α without the plasmid, were obtained by inoculation of 5 ml of LB in 15 ml Falcon tubes with colonies from agar plates and incubation overnight. From these starter cultures 50 ml of M9 supplemented minimal medium in 500 ml Erlenmayer flasks were inoculated to an OD at 600 nm (path length 1 cm) of 0.05 to obtain precultures. These precultures were incubated until an OD at 600 nm (path length 1 cm) of 0.99 for E. coli DH5α harboring pSEVA132_BBa_K082034 and 1.366 for E. coli DH5α without the plasmid respectively. The cultures for the measurements were prepared by inoculating 10 ml of minimal medium in 100 ml Erlenmayer flasks with the precultures to an OD at 600 nm (path length 1 cm) of approximately 0.05. Optical Density at 595 nm and fluorescence (excitation at 485 nm and emission at 530 nm) were measured in a microtiter plate with 200 μl sample using a PerkinElmer Victor3 Fluorometer.

Results
E. coli DH5α harboring pSEVA132_BBa_K082034, minimal medium, induction at 180 min.
Cell density of E. coli DH5α harboring pSEVA132_BBa_K082034 over time with different inducer concentrations.
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.
E. coli DH5α without pSEVA132_BBa_K082034, minimal medium, induction at 180 min.
Cell density of E. coli DH5α harboring pSEVA132_BBa_K082034 over time.
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.

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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