Difference between revisions of "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 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

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.	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.
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 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.]

User Reviews

UNIQ4074e0b064c24207-partinfo-00000001-QINU UNIQ4074e0b064c24207-partinfo-00000002-QINU

@@ Line 44: / Line 44: @@
 <br>
-====Expression Behaviour of BBa_K082034 in pSB1A2====
-=====Methods=====
-[[Image:PSB1A2.png|thumb|right|300px|'''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.
-<br>The measured fluorescence and optical density was corrected by an LB blank. The corrected value of the fluorescence was divided by the optical density to obtain fluorescence per cell density. Normalization by the control (''E. coli'' DH5α cells not carrying the plasmid) resulted in the graph found to the right.
-=====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 level of BBa_K082034 present in pSB1A2_BBa_K082034 harboring cells. Thus, the fluorescence observed resulted from "leaky" expression, while the effect of the inducer was probably hidden by noise. The experiment would need to be repeated in order to test for reproducibility. Background fluorescence could be minimized by using M9 supplemented minimal medium.
 ====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, 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.
+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=====