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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.

Conclusion

BBa_K082034 seems to be present in such high numbers that the chromosomally encoded LacI cannot repress it sufficiently anymore. pSEVA132 is thus not a good choice if inducible fluorescence is required.

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

UNIQ1e119ded10e35446-partinfo-00000001-QINU UNIQ1e119ded10e35446-partinfo-00000002-QINU

@@ Line 6: / Line 6: @@
 ====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).
+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====
 {| border="1" align="left"
 |+
-! plasmid !! origin !! resistance !! additional information
+! plasmid !! purpose !! origin !! resistance !! additional information
 |-
-| pSB1A2 || pMB1; 100-300 copies/cell || amp || [https://parts.igem.org/wiki/index.php?title=Part:pSB1A2 link to registry]
+| pSEVA132 || expression of BBa_K082034 || pBBR1; approx. 75 copies/cell || kan || Victor de Lorenzo's lab; [https://parts.igem.org/Part:BBa_K082034:Experience#Reference analysis of copy number] (pSEVA132 = wv1)
 |-
-| 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 || expression of lacI || pBR322; high copy ||  tet, amp || [1]
-|-
-| pKQV4 || pBR322 ||  tet, amp || [1]; contains lacIq gene
 |-
 |}
@@ Line 26: / Line 24: @@
 <br>
 <br>
 ====Cloning====
 {| border="0" align="right"
@@ Line 32: / Line 31: @@
 |[[Image:Control digest pSEVA132.png|120px|thumb| 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.
+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.
-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.
 <br>
 <br>
 '''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.
 <br>
-'''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.
+'''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.
 <br>
 <br>
@@ Line 46: / Line 44: @@
 <br>
-====pSB1A2_BBa_K082034====
-=====Methods=====
+====Expression Behaviour of BBa_K082034 in pSEVA132====
-[[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 harboring (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.
-<br>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 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.
-====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, 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.
-<br>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=====
+pSEVA132_BBa_K082034, induction at 60 min.
 {| border="0" align="center"
@@ Line 73: / Line 60: @@
 |[[Image:PSEVA%2C_induced%2C_fl.png|260px|thumb| Fluorescence of ''E. coli'' DH5α harboring pSEVA132_BBa_K082034 over time. Induction at 60 min.]]
 |[[Image:PSEVA%2C_induced%2C_fl_OD.png|260px|thumb| Fluorescence per cell density over time of ''E. coli'' DH5α harboring pSEVA132_BBa_K082034. Induction at 60 min.]]
+|}
+pSEVA132_BBa_K082034, no induction.
+{| border="0" align="center"
 |-
-|[[Image:PSEVA%2C_not_induced%2C_OD.png|260px|thumb| Cell density of ''E. coli'' DH5α harboring pSEVA132_BBa_K082034 over time. No induction.]]
+|[[Image:PSEVA%2C_not_induced%2C_OD.png|260px|thumb| Cell density of ''E. coli'' DH5α harboring pSEVA132_BBa_K082034 over time.]]
 |[[Image:PSEVA%2C_not_induced%2C_fl.png|260px|thumb| Fluorescence of ''E. coli'' DH5α harboring pSEVA132_BBa_K082034 over time. No induction.]]
 |[[Image:PSEVA%2C_not_induced%2C_fl_OD1.png|260px|thumb| 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.
+{| border="0" align="center"
 |-
 |[[Image:PSEVApKQV%2C_induced%2C_OD.png|260px|thumb| Cell density of ''E. coli'' DH5α harboring pSEVA132_BBa_K082034 and pKQV4_lacIq over time.]]
@@ Line 84: / Line 78: @@
 =====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.
+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.
 <br>
-For constitutive expression of GFP the system with pSEVA132_BBa_K082034 could be useful.
+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.
+<br>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).
+<br>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.
+<br>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.
+{| border="0" align="center"
+|-
+|[[Image:Exp od.png|260px|thumb| Cell density of ''E. coli'' DH5α harboring pSEVA132_BBa_K082034 over time with different inducer concentrations.]]
+|[[Image:Exp fl.png|260px|thumb| Fluorescence of ''E. coli'' DH5α harboring pSEVA132_BBa_K082034 over time. Induction at 60 min.]]
+|[[Image:Exp fl od.png|260px|thumb| 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.
+{| border="0" align="center"
+|-
+|[[Image:control od.png|260px|thumb| Cell density of ''E. coli'' DH5α harboring pSEVA132_BBa_K082034 over time.]]
+|[[Image:control fl.png|260px|thumb| Fluorescence of ''E. coli'' DH5α harboring pSEVA132_BBa_K082034 over time. No induction.]]
+|[[Image:control fl od.png|260px|thumb| Fluorescence per cell density over time of ''E. coli'' DH5α harboring pSEVA132_BBa_K082034. No induction.]]
+|}
+=====Conclusion=====
+BBa_K082034 seems to be present in such high numbers that the chromosomally encoded LacI cannot repress it sufficiently anymore. pSEVA132 is thus not a good choice if inducible fluorescence is required.
 ====Reference====