Team:UNIPV-Pavia/New Parts

From 2009.igem.org

(Difference between revisions)

Latest revision as of 23:11, 21 October 2009

Our new parts

Our new parts:

Re-built existing parts (BBa_our part code/BBa_existing part code)

Existing parts from the Registry

Existing parts: sequence debugging

BBa_K173003 - ethanol producing device

Description

This device takes PoPS as input and produces pyruvate decarboxylase (pdc) and alcohol dehydrogenase II (adhB) enzymes.

Pyruvate decarboxylase (pdc, ) catalyses the decarboxylation of pyruvic acid to acetaldehyde and carbon dioxide, while alcohol dehydrogenase II (adhB, ) catalyses the acetaldehyde reduction to ethanol. The latter enzyme, as reported in the literature, is also able to work in the opposite direction, oxidizing ethanol to acetaldehyde. The two enzymes come from Zymomonas mobilis ethanologenic bacterium and constitute an essential step of alcoholic fermentation.

So, this device contains the minimum set of genes that are required to engineer a heterologous fermentation pathway.

The coding sequences of pdc and adhB genes have been optimized for Escherichia coli codon usage.

Characterization

Qualitative phenotype characterization

PLATES RESULTS

This device has been cloned downstream of 4 different promoters, one of which in two different vectors. These assemblies were transformed in TOP10 and gave the following phenotypes when bacteria were plated on LB agar plates or on LB agar plates + 2% glucose (all the plates have been incubated at 37°C for about 11 hours, then some colonies were picked with a sterile tip and plates were incubated for additional 10 hours):

upstream promoter	plasmid	promoter RPU in LB	colony phenotype
		1.58 [1.56; 1.67]	small colonies
		not estimated	small colonies
		0.69 [0.64; 0.73]	small colonies
		~0 without induction	normal colonies
		not estimated	normal colonies

These results demonstrate that a constitutive expression of pdc and adhB gives a high metabolic burden to E. coli, in fact the only normal phenotype on plates was with non-induced device. Only this assembled part has been submitted to the Registry, as .

BBa_F2620-BBa_K173003 in pSB1AK3 plate: example of normal size colonies.

BBa_K173005-BBa_K173003 in pSB1AK3 plate: example of small size colonies.

LIQUID CULTURES RESULTS

Grown cultures of and after the FIRST INOCULUM showed different phenotypes (qualitative analysis), as a function of the used protocol. was the regulated operon, was the promoterless operon, was the promoter alone, pSB1AK3 was a high copy plasmid and pSB4C5 was a low copy plasmid.

PROTOCOL#1 RESULTS (i.e. shaken anaerobic falcon tubes, no glucose):

BioBrick	plasmid	induction with 3OC6-HSL	phenotype
	pSB1AK3	no	normal growth
	pSB1AK3	no	normal growth
	pSB4C5	no	normal growth
BBa_F2620 ALONE - negative control	pSB1A2	no	normal growth

PROTOCOL#2 RESULTS (i.e. unshaken anaerobic falcon tubes, 2% glucose):

BioBrick	plasmid	induction with 3OC6-HSL	phenotype
	pSB1AK3	no	a lot of foam in the upper side of the culture; a strangely big and cloudy pellet after 4000 rpm centrifugation.
	pSB1AK3	100 nM	no growth
	pSB1AK3	1 uM	no growth
BBa_F2620 ALONE - negative control	pSB1A2	no	normal growth
BBa_F2620 ALONE - negative control	pSB1A2	100 nM	normal growth
BBa_F2620 ALONE - negative control	pSB1A2	1 uM	normal growth
	pSB1AK3	no	normal growth

PROTOCOL#3 RESULTS (i.e. shaken anaerobic falcon tubes, 2% glucose):

BioBrick	plasmid	induction with 3OC6-HSL	phenotype
	pSB1AK3	no	higher amount of bacterial pellet than in the negative control (BBa_F2620) after 4000 rpm centrifugation
	pSB1AK3	100 nM	no growth
	pSB1AK3	1 uM	no growth
	pSB4C5	no	a bit higher amount of bacterial pellet than in the negative control (BBa_F2620), but lower than in pSB1AK3 after 4000 rpm centrifugation
	pSB4C5	1 uM	a bit higher amount of bacterial pellet than in the negative control (BBa_F2620), but lower than in pSB1AK3 after 4000 rpm after 4000 rpm centrifugation
BBa_F2620 ALONE - negative control	pSB1A2	no	normal growth
BBa_F2620 ALONE - negative control	pSB1A2	100 nM	normal growth
BBa_F2620 ALONE - negative control	pSB1A2	1 uM	normal growth
	pSB1AK3	no	normal growth

These results confirm the metabolic burden given by this BioBrick when gene expression is triggered: induced strains bearing a high copy plasmid containing do not survive, while low copy ones can survive even at 1 uM HSL, which is a high induction, according to the characterization results on (see its own page on the Registry and our our characterization experiments on it). We decided to exploit the lux promoter leakage activity for in pSB1AK3 (high copy), while we decided to induce in pSB4C5 (low copy) the with 1 uM of 3OC6-HSL during our fermentation experiments.

The qualitative phenotypes of the grown cultures of and after fermentation in 10% glucose showed that pellets were higher in (in both pSB1AK3 uninduced and pSB4C5 induced) than in and in the negative control .

Moreover, after both inoculum steps, the grown cultures of and (in both pSB1AK3 uninduced and pSB4C5 induced) showed a "sparkling" phenotype, while and in the negative control did not.

Quantitative characterization

1 - Growth of strains bearing:

3OC6-HSL inducible system upstream of the operon in high copy plasmid () uninduced;
3OC6-HSL inducible system upstream of the operon in low copy plasmid () uninduced;
3OC6-HSL inducible system upstream of the operon in low copy plasmid () induced with 1 uM of 3OC6-HSL;
3OC6-HSL inducible system without the operon in high copy plasmid ();
a promoterless operon in high copy plasmid ().

has been monitored in selective LB + 10% glucose starting from an inoculum in the microplate reader (figure below).

Protocol: 200 ul aliquots (3 replicates) were taken from a just inoculated 30 ml culture (LB + 10% glucose) of the SECOND INOCULUM of PROTOCOL#3 and let grow in the microplate reader according to the automatic protocol described in Microplate reader experiments. The microplate was "sealed" with parafilm in order to create an anaerobic condition.

Mean values on three wells filled with aliquots of the indicated strains (200 ul) in selective LB + 10% glucose.

Mean values with 95% confidence intervals for the mean on three wells filled with aliquots of the indicated strains (200 ul) in selective LB + 10% glucose.

2 - Ethanol fermentation capability of these strains in 10%w/v glucose-supplemented LB medium has been tested with three different fermentation protocols (see Fermentation experiments for a detailed description).

The main fermentation results can be summarized with the following bar plots. Ethanol and pH have been measured after 48 hours ( PROTOCOL#2, first two measurements) or 24 hours ( PROTOCOL#3, other measurements).

Ethanol production after fermentation of 10% glucose.

pH after fermentation of 10% glucose.

PROTOCOL#1 was tested only on (in pSB1AK3 induced with 100 nM HSL) and as a negetive control. This protocol resulted in bacterial death for (in pSB1AK3 induced with 100 nM HSL) because of the previously explained metabolic burden when pdc and adhB gene expression was triggered at high levels in a high copy plasmid.

Comments:

The pH results suggest that in strains bearing expressed pdc and adhB the fermentation does not give high levels of organic acids as in negative control strains which do not have pdc and adhB. It is surprising that the pH of BBa_K173003 in pSB1AK3 (pdc and adhB without any promoter) is higher than in the negative control. This could be due to a weak spurious transcription of pdc and adhB, amplified by the high copy number plasmid. This may re-direct part of pyruvate metabolism to ethanol and not to organic acids. Of course this phenomenon should be further investigated.

The results about dynamic growth (in terms of O.D.600) are in accordance with the pH results, confirming that strains bearing BBa_K173021 in pSB1AK3 produce less organic acids than the negative controls, hopefully thanks to pdc and adhB which re-direct the metabolism of pyruvate from organic acids to ethanol, and consequently grow better (to OD600=~1). No significant difference was noticed in the growth of promoterless operon, uninduced and induced BBa_K173021 in pSB4C5, suggesting that in promoterless operon transcription could be activated in unspecific manner (i.e. without a promoter upstream). These three cultures reached OD600 values of ~0.7, while the negative control () reached only OD600=~0.4.

Fermentation results showed that strains bearing expressed pdc and adhB in pSB1AK3 high copy plasmid can produce less ethanol than the negative controls in both PROTOCOL#2 and PROTOCOL#3. Our hypothesis is that in our working conditions adhB converts part of the fermented ethanol back to acetaldehyde, in fact this enzyme is able to perform both acetaldehyde conversion to ethanol and the opposite reaction.

On the other hand, if we add 1%w/v of ethanol in the medium this strain can ferment 30 g/l of ethanol, which is about 60% of theoretical yield. This may be due to adhB behaviour, which was reported in literature to be activated by ethanol accumulation.

Moreover, It is possible that when starting from less than 1% of ethanol in fermentation medium ethanol production could be increased, because in our experiment the bacterial culture reached ethanol concentration survival threshold (40 g/l) and so it difficult to ferment more than 30 g/l.

Conclusions

Ethanol production in this operon has been proved for when assembled under the control of HSL receiver device, yielding composite part.

Promising working conditions have been found for it:

BBa_K173021 in pSB1AK3 (HC) not induced with 3OC6-HSL (exploiting basic transcriptional activity), induced with 1%w/v of ethanol;
BBa_K173021 in pSB4C5 (LC) induced with 1 uM of 3OC6-HSL.

Increased pH and growth in ethanol producing strains are also proves that the pyruvate metabolism is re-directed from organic acid fermentation to ethanol production. Further work has to be performed to fully understand pdc and adhB optimal working conditions and to optimize the environment for a high yield ethanol production.

Top

BBa_K173007 - aTc inducible device with J23100 promoter

Description

This is an aTc sensing device.

promoter drives the constitutive production of tetR repressor (), which inhibits tetR promoter () activity. When aTc is added to the medium, it binds tetR and inhibits it. So, the PoPS output is a function of the aTc concentration.

A tight regulation is expected for this inducible system because BBa_J23100 is a strong promoter and so tetR repressor should be produced at extremely high levels.

The data below are referred to , which is the measurement system of .

Characterization

aTc concentration [ng/ml]	LB		M9 supplemented
aTc concentration [ng/ml]	Doubling time [minutes]	RPU mean [range]	Doubling time [minutes]	RPU mean [range]
0	36	0.05 [~0 ; 0.15]	59	0.03 [ 0.02 ; 0.04]
25	32	0.12 [~0 ; 0.19]	61	0.03 [ 0.02 ; 0.04]
50	34	0.17 [0.05; 0.25]	57	0.04 [ 0.03 ; 0.05]
75	43	0.15 [~0; 0.23]	65	0.03 [ 0.02 ; 0.06]
100	40	0.21 [0.19; 0.23]	62	0.03 [ 0.03 ; 0.05]
200	40	0.25 [0.20; 0.28]	70	0.04 [ 0.03 ; 0.06]
300	43	0.20 [0.18; 0.21]	67	0.05 [ 0.04 ; 0.08]

Growth curves for BBa_K173009 in M9

Growth curves for BBa_K173009 in LB

(dGFP/dt)/OD in LB

(dGFP/dt)/OD in M9

Induction curve of BBa_K173009 in M9

Induction curve of BBa_K173009 in LB

Conclusions

We demonstrated that this part works as expected, sensing the aTc concentration provided in the culture medium. The transfer function of this sensor has been characterized in standard units (RPUs) in two different growth media (LB and M9 supplemented with glycerol), as well as the metabolic burden (in terms of doubling time) which affects E. coli bearing this part.

On the other hand, we did not expect to have a higher GFP synthesis rate per cell after the exponential growth phase than in the exponential phase itself (as reported in the 3rd plot for both LB and M9).

This part shows to have a very low leakage rate (about 0.025 RPU) but also a very low transcription rate for high concentrations of aTc. So, it can be used for tight regulation, in those systems who need very low leakage rates (no gene expressed in absence of inducer) but not for massive protein production.

Unfortunately, the estimated transfer function is very noisy (probably because the measured fluorescence values are very close to blank values), and further experiments should be performed in order to validate these induction curves.

Top

BBa_K173011 - aTc inducible device with J23118 promoter

Description

This is an aTc sensing device.

promoter drives the constitutive production of tetR repressor (), which inhibits tetR promoter () activity. When aTc is added to the medium, it binds tetR and inhibits it. So, the PoPS output is a function of the aTc concentration.

A less tight regulation is expected for this inducible system than in because BBa_J23118 promoter is weaker than BBa_J23100 and so tetR repressor shold be produced at lower levels than in the other sensor.

The data below are referred to , which is the measurement system of .

Characterization

aTc concentration [ng/ml]	LB		M9 supplemented
aTc concentration [ng/ml]	Doubling time [minutes]	RPU	Doubling time [minutes]	RPU
0	36	0.51 [0.43; 0.73]	78	0.96 [0.80; 1.23]
25	33	1.36 [1.28; 1.44]	82	1.66 [1.38; 2.11]
50	40	1.39 [1.30; 1.48]	81	1.85 [1.61; 2.27]
75	43	1.25 [1.17; 1.31]	88	1.75 [1.51; 2.15]
100	43	1.40 [1.28; 1.48]	90	1.89 [1.68; 2.34]
200	45	1.37 [ 1.31 ; 1.41]	92	1.75 [1.51; 2.09]
300	48	1.45 [ 1.36 ; 1.55]	94	1.73 [1.53; 2.07]

Growth curves for BBa_K173026 in M9

Growth curves for BBa_K173026 in LB

(dGFP/dt)/OD in LB and M9

Induction curve of BBa_K173026 in M9

Induction curve of BBa_K173026 in LB

Conclusions

We demonstrated that this part works as expected because GFP is produced as an increasing function of the aTc concentration provided in the culture medium. The transfer function of this sensor has been characterized in standard units (RPUs) in two different growth media (LB and M9 supplemented with glycerol), as well as the metabolic burden (in terms of doubling time) which affects E. coli bearing this part.

On the other hand, as for BBa_K173007, we did not expect to have a higher GFP synthesis rate per cell after the exponential growth phase than in the exponential phase itself (as reported in the 3rd plot).

This part shows a high level of leakage (about 0.5 RPU in LB and 1 RPU in M9) but also high levels of induction for high concentrations of aTc (about 1.45 RPU in LB and 1.7 RPU in M9). For this reason, it can be used in those cases where an important response in terms of gene expression is required in presence of an inducer, but is not suitabe if the absence of expression has to be off when inducer is absent. Its induction range is 0.95 RPU in LB and 0.7 RPU in M9.

The switch point of this device is between 0 ng/ml and 25 ng/ml of aTc in both LB and M9.

Top

BBa_K173010 - lactose/IPTG inducible device with J23118 promoter

Description

This should work as a lactose/IPTG sensor.

promoter drives the constitutive production of lacI repressor (), which inhibits lac promoter () activity. When lactose or IPTG is added to the medium, it binds lacI and inhibits it. So, the PoPS output is a function of lactose/IPTG concentration.

Thanks to the hybrid lac promoter (), designed taking the Plambda promoter () and substituting its cI () binding sites with two lacI binding sites, the behaviour of this device is not a function of glucose concentration because the wild type CAP binding sites are not present in this artificial lac promoter.

The test performed to characterize this device has been done with , which is the measurement system of itself.

Characterization

We inoculated 8 ul of glycerol stock in 5 ml of LB + Amp and incubated it overnight (37°C, 220 rpm).
In the morning we diluted 1:100 the culture in two falcon tubes and incubated them under the same conditions as before.
After about 6 hours we added IPTG in one of the two cultures (final concentration = 1 mM).
Fluorescence and O.D.600 were measured in the microplate reader after additional 4 hours.

Results: both cultures had a comparable O.D.600, but also had the same RFU values, demonstrating that the induced culture did not produce any GFP.

Conclusions

We did not perform any standard measurement on this device because preliminary tests showed that GFP levels of induced and non induced cultures were the same and were equals to negative control.

Unfortunately, we did not check if the sequence of , so we do not know if it was actually correct. Another possibility is that lacI is produced at so high levels that 2 mM of IPTG is not sufficient to induce the lac promoter.

Further tests should be done for this system.

Top

@@ Line 30: / Line 30: @@
 *[[Team:UNIPV-Pavia/New_Parts#BBa_K173010 - lactose/IPTG inducible device with J23118 promoter| BBa_K173010 - lactose/IPTG inducible device with J23118 promoter]]
-[[Team:UNIPV-Pavia/Rebuilt#Re-built existing parts| '''Re-built existing parts (BBa_our part code/BBa_existing part code)''':]]
+[[Team:UNIPV-Pavia/Rebuilt| '''Re-built existing parts (BBa_our part code/BBa_existing part code)''']]
-[[Team:UNIPV-Pavia/Existing#Existing parts from the Registry| '''Existing parts from the Registry''']]
+[[Team:UNIPV-Pavia/Existing| '''Existing parts from the Registry''']]
-[[Team:UNIPV-Pavia/Debug#Existing parts: sequence debugging| '''Existing parts: sequence debugging''']]
+[[Team:UNIPV-Pavia/Debug| '''Existing parts: sequence debugging''']]
 __NOTOC__
@@ Line 61: / Line 61: @@
 ''PLATES RESULTS''
-This device has been cloned downstream of 4 different promoters, one of which in two different vectors. These assemblies were transformed in TOP10 and gave the following phenotypes on LB agar plates and also on LB agar plates + 2% glucose (all the plates have been incubated at 37°C for about 11 hours, then some colonies were picked with a sterile tip and plates were incubated for additional 10 hours):
+This device has been cloned downstream of 4 different promoters, one of which in two different vectors. These assemblies were transformed in TOP10 and gave the following phenotypes when bacteria were plated on LB agar plates or on LB agar plates + 2% glucose (all the plates have been incubated at 37°C for about 11 hours, then some colonies were picked with a sterile tip and plates were incubated for additional 10 hours):
 {|align="center" border=1
-|'''promoter upstream of <partinfo>BBa_K173003</partinfo>'''
+|'''upstream promoter'''
 |'''plasmid'''
 |'''promoter RPU in LB'''
@@ Line 70: / Line 70: @@
 |<partinfo>BBa_K173005</partinfo>
 |<partinfo>pSB1AK3</partinfo>
-|?
+|1.58 [1.56; 1.67]
 |small colonies
 |-
@@ Line 80: / Line 80: @@
 |<partinfo>BBa_J23118</partinfo>
 |<partinfo>pSB1A2</partinfo>
-|?
+|0.69 [0.64; 0.73]
 |small colonies
 |-
 |<partinfo>BBa_F2620</partinfo>
 |<partinfo>pSB1AK3</partinfo>
-|? without induction
+|~0 without induction
-|normal size colonies
+|normal colonies
 |-
 |<partinfo>BBa_F2620</partinfo>
 |<partinfo>pSB4C5</partinfo>
 |not estimated
-|normal size colonies
+|normal colonies
 |}
-These results demonstrate that a constitutive expression of pdc and adhB gives a high metabolic burden to E. coli, in fact the only normal phenotype on plates was <partinfo>BBa_K173003</partinfo> with <partinfo>BBa_F2620</partinfo> non-induced device. This assembled part has been submitted to the Registry as <partinfo>BBa_K173021</partinfo>.
+These results demonstrate that a constitutive expression of pdc and adhB gives a high metabolic burden to E. coli, in fact the only normal phenotype on plates was <partinfo>BBa_K173003</partinfo> with <partinfo>BBa_F2620</partinfo> non-induced device. Only this assembled part has been submitted to the Registry, as <partinfo>BBa_K173021</partinfo>.
 {|align="center"
@@ Line 104: / Line 104: @@
 ''LIQUID CULTURES RESULTS''
-Grown cultures of <partinfo>BBa_K173003</partinfo> and <partinfo>BBa_K173021</partinfo> after the FIRST INOCULUM showed different phenotypes (qualitative analysis), as a function of the used protocol.
+Grown cultures of <partinfo>BBa_K173003</partinfo> and <partinfo>BBa_K173021</partinfo> after the FIRST INOCULUM showed different phenotypes (qualitative analysis), as a function of the used protocol. <partinfo>BBa_K173021</partinfo> was the regulated operon, <partinfo>BBa_K173003</partinfo> was the promoterless operon, <partinfo>BBa_F2620</partinfo> was the promoter alone, pSB1AK3 was a high copy plasmid and pSB4C5 was a low copy plasmid.
@@ Line 211: / Line 211: @@
 |pSB4C5
 |1 uM
-|a bit higher amount of bacterial pellet than in the negative control (BBa_F2620),<br>but lower than in pSB1AK3 after 4000 rpm after 4000 rpm centrifugationcentrifugation
+|a bit higher amount of bacterial pellet than in the negative control (BBa_F2620),<br>but lower than in pSB1AK3 after 4000 rpm after 4000 rpm centrifugation
 |-
 |BBa_F2620 ALONE<br>- negative control
@@ Line 234: / Line 234: @@
 |}
-These results confirm the metabolic burden given by this BioBrick when gene expression is triggered: induced strains bearing a high copy plasmid containing <partinfo>BBa_K173021</partinfo> do not survive, while low copy ones can survive even at high induction (1 uM). We decided to exploit the lux promoter leakage activity for <partinfo>BBa_K173021</partinfo> in pSB1AK3 (high copy), while we decided to induce <partinfo>BBa_K173021</partinfo> in pSB4C5 (low copy) the with 1 uM of 3OC6-HSL during our fermentation experiments.
+These results confirm the metabolic burden given by this BioBrick when gene expression is triggered: induced strains bearing a high copy plasmid containing <partinfo>BBa_K173021</partinfo> do not survive, while low copy ones can survive even at 1 uM HSL, which is a high induction, according to the characterization results on <partinfo>BBa_F2620</partinfo> (see its own page on the Registry and our [https://2009.igem.org/Team:UNIPV-Pavia/Existing#BBa_F2620_-_3OC6HSL_receiver_device our characterization experiments on it]). We decided to exploit the lux promoter leakage activity for <partinfo>BBa_K173021</partinfo> in pSB1AK3 (high copy), while we decided to induce <partinfo>BBa_K173021</partinfo> in pSB4C5 (low copy) the with 1 uM of 3OC6-HSL during our fermentation experiments.
 The qualitative phenotypes of the grown cultures of <partinfo>BBa_K173003</partinfo> and <partinfo>BBa_K173021</partinfo> after fermentation in 10% glucose showed that pellets were higher in <partinfo>BBa_K173021</partinfo> (in both pSB1AK3 uninduced and pSB4C5 induced) than in <partinfo>BBa_K173003</partinfo> and in the negative control <partinfo>BBa_F2620</partinfo>.
@@ Line 245: / Line 245: @@
 '''Quantitative characterization'''
-''pH''
+- Growth of strains bearing:
+*3OC6-HSL inducible system upstream of the operon in high copy plasmid (<partinfo>BBa_K173021</partinfo>) uninduced;
+*3OC6-HSL inducible system upstream of the operon in low copy plasmid (<partinfo>BBa_K173021</partinfo>) uninduced;
+*3OC6-HSL inducible system upstream of the operon in low copy plasmid (<partinfo>BBa_K173021</partinfo>) induced with 1 uM of 3OC6-HSL;
+*3OC6-HSL inducible system without the operon in high copy plasmid (<partinfo>BBa_F2620</partinfo>);
+*a promoterless operon in high copy plasmid (<partinfo>BBa_K173003</partinfo>).
-pH has been measured in grown cultures after the FIRST INOCULUM (in 2% glucose after 24 hours) and after the SECOND INOCULUM (in 10% glucose after 24 or 48 hours). The measurements performed are the following:
+has been monitored in selective LB + 10% glucose starting from an inoculum in the microplate reader (figure below).
-[[Image:pv_ph_1.jpg|PROTOCOL#1: pH at the end of fermentation (after the SECOND INOCULUM).]]
+''Protocol:'' 200 ul aliquots (3 replicates) were taken from a just inoculated 30 ml culture (LB + 10% glucose) of the SECOND INOCULUM of [[Team:UNIPV-Pavia/Parts_Characterization#PROTOCOL#3| PROTOCOL#3]] and let grow in the microplate reader according to the automatic protocol described in [[Team:UNIPV-Pavia/Parts_Characterization#Microplate reader experiments| Microplate reader experiments]]. The microplate was "sealed" with parafilm in order to create an anaerobic condition.
-[[Image:pv_ph_2.jpg|PROTOCOL#2: pH after the FIRST and the SECOND (end of fermentation) INOCULUM]]
+{|align="center"
+|[[Image:pv_OD_ethanol.jpg|thumb|500px|Mean values on three wells filled with aliquots of the indicated strains (200 ul) in selective LB + 10% glucose.]]
+|}
-[[Image:pv_ph_3.jpg|PROTOCOL#3: pH after the FIRST and the SECOND (end of fermentation) INOCULUM]]
+{|align="center"
+|[[Image:pv_OD_ethanol_err.jpg|thumb|500px|Mean values with 95% confidence intervals for the mean on three wells filled with aliquots of the indicated strains (200 ul) in selective LB + 10% glucose.]]
+|}
-The shown results suggest that in strains bearing expressed pdc and adhB the fermentation does not give high levels of organic acids as in negative control strains which do not have pdc and adhB. It is surprising that the pH of <partinfo>BBa_K173003</partinfo> in pSB1AK3 (pdc and adhB without any promoter) is slightly higher than in the negative controls. This could be due to a weak spurious transcription of pdc and adhB, amplified by the high copy number plasmid. This may re-direct part of pyruvate metabolism to ethanol and not to organic acids. Of course this phenomenon should be further investigated.
+- Ethanol fermentation capability of these strains in 10%w/v glucose-supplemented LB medium has been tested with three different fermentation protocols (see [[Team:UNIPV-Pavia/Parts_Characterization#Fermentation_experiments| Fermentation experiments]] for a detailed description).
-''Growth curves''
+The main fermentation results can be summarized with the following bar plots. Ethanol and pH have been measured after 48 hours ([[Team:UNIPV-Pavia/Parts_Characterization#PROTOCOL.232| PROTOCOL#2]], first two measurements) or 24 hours ([[Team:UNIPV-Pavia/Parts_Characterization#PROTOCOL.233| PROTOCOL#3]], other measurements).
-<partinfo>BBa_K173021</partinfo> and its suitable negative controls growth was tested in our microplate reader: 200 ul aliquots (3 or 4 replicates) were taken from a just inoculated 30 ml culture (LB + 10% glucose) of the SECOND INOCULUM of [[Team:UNIPV-Pavia/Parts_Characterization#PROTOCOL#3| PROTOCOL#3]] and let grow in the microplate reader according to the automatic protocol described in [[Team:UNIPV-Pavia/Parts_Characterization#Microplate reader experiments| Microplate reader experiments]]. The microplate was "sealed" with parafilm in order to create an anaerobic condition.
+{|align="center"
+|[[Image:pv_ethanol_results_bar.jpg|500px|thumb|Ethanol production after fermentation of 10% glucose.]]
+|}
-[[Image:Pv_OD_ethanol.jpg|500px|Growth curves of <partinfo>BBa_K173003</partinfo>, <partinfo>BBa_K173021</partinfo> in pSB1AK3, <partinfo>BBa_K173021</partinfo> in pSB4C5 + 1uM HSL, <partinfo>BBa_F2620</partinfo> in pSB1A2 negative control, <partinfo>BBa_B0015</partinfo> in pSB1AK3 negative control]]
+{|align="center"
+|[[Image:pv_ethanol_results_ph.jpg|500px|thumb|pH after fermentation of 10% glucose.]]
+|}
-[[Image:Pv_OD_ethanol_err.jpg|500px|Mean value and 95% confidence intervals for the growth curves.]]
+[[Team:UNIPV-Pavia/Parts_Characterization#PROTOCOL.232| PROTOCOL#1]] was tested only on <partinfo>BBa_K173003</partinfo> (in pSB1AK3 induced with 100 nM HSL) and <partinfo>BBa_F2620</partinfo> as a negetive control. This protocol resulted in bacterial death for <partinfo>BBa_K173003</partinfo> (in pSB1AK3 induced with 100 nM HSL) because of the previously explained metabolic burden when pdc and adhB gene expression was triggered at high levels in a high copy plasmid.
-This test showed that <partinfo>BBa_K173021</partinfo> in pSB1AK3 is able to reach much higher OD600 than the other cultures, reaching ? values. <partinfo>BBa_K173021</partinfo> in pSB4C5 both induced and uninduced had comparable growth curves and reached OD600 of about ?. Negative controls (<partinfo>BBa_F2620</partinfo> in pSB1A2 and <partinfo>BBa_B0015</partinfo> in pSB1AK3) had comparable growth curves and reached low OD (about ?). It is surprising that <partinfo>BBa_K173003</partinfo> in pSB1AK3 reached a higher OD (?) than the negative controls.
-The shown results are in complete accordance with the pH results, confirming that strains bearing <partinfo>BBa_K173021</partinfo> in pSB1AK3 and in pSB4C5 produce less organic acids than the negative controls, hopefully thanks to pdc and adhB and consequently grow better.
+''Comments:''
+The pH results suggest that in strains bearing expressed pdc and adhB the fermentation does not give high levels of organic acids as in negative control strains which do not have pdc and adhB. It is surprising that the pH of BBa_K173003 in pSB1AK3 (pdc and adhB without any promoter) is higher than in the negative control. This could be due to a weak spurious transcription of pdc and adhB, amplified by the high copy number plasmid. This may re-direct part of pyruvate metabolism to ethanol and not to organic acids. Of course this phenomenon should be further investigated.
-''Ethanol production''
+The results about dynamic growth (in terms of O.D.600) are in accordance with the pH results, confirming that strains bearing BBa_K173021 in pSB1AK3 produce less organic acids than the negative controls, hopefully thanks to pdc and adhB which re-direct the metabolism of pyruvate from organic acids to ethanol, and consequently grow better (to OD600=~1). No significant difference was noticed in the growth of promoterless operon, uninduced and induced BBa_K173021 in pSB4C5, suggesting that in promoterless operon transcription could be activated in unspecific manner (i.e. without a promoter upstream). These three cultures reached OD600 values of ~0.7, while the negative control (<partinfo>BBa_F2620</partinfo>) reached only OD600=~0.4.
-Here we report the fermentation results of 100 g/l of glucose:
-[[Image:pv_etoh_1.jpg|PROTOCOL#1: ethanol at the end of fermentation.]]
+Fermentation results showed that strains bearing expressed pdc and adhB in pSB1AK3 high copy plasmid can produce less ethanol than the negative controls in both [[Team:UNIPV-Pavia/Parts_Characterization#PROTOCOL.232| PROTOCOL#2]] and [[Team:UNIPV-Pavia/Parts_Characterization#PROTOCOL.233| PROTOCOL#3]]. Our hypothesis is that in our working conditions adhB converts part of the fermented ethanol back to acetaldehyde, in fact this enzyme is able to perform both acetaldehyde conversion to ethanol and the opposite reaction.
-[[Image:pv_etoh_2.jpg|PROTOCOL#2: ethanol at the end of fermentation.]]
+On the other hand, if we add 1%w/v of ethanol in the medium this strain can ferment 30 g/l of ethanol, which is about 60% of theoretical yield. This may be due to adhB behaviour, which was reported in literature to be activated by ethanol accumulation.
-[[Image:pv_etoh_2.jpg|PROTOCOL#2: ethanol at the end of fermentation.]]
+Moreover, It is possible that when starting from less than 1% of ethanol in fermentation medium ethanol production could be increased, because in our experiment the bacterial culture reached ethanol concentration survival threshold (40 g/l) and so it difficult to ferment more than 30 g/l.
-[[Image:pv_etoh_3.jpg|PROTOCOL#3: ethanol at the end of fermentation.]]
 ===Conclusions===
+Ethanol production in this operon has been proved for <partinfo>BBa_K173003</partinfo> when assembled under the control of <partinfo>BBa_F2620</partinfo> HSL receiver device, yielding <partinfo>BBa_K173021</partinfo> composite part.
+Promising working conditions have been found for it:
+*BBa_K173021 in pSB1AK3 (HC) not induced with 3OC6-HSL (exploiting <partinfo>BBa_F2620</partinfo> basic transcriptional activity), induced with 1%w/v of ethanol;
+*BBa_K173021 in pSB4C5 (LC) induced with 1 uM of 3OC6-HSL.
+Increased pH and growth in ethanol producing strains are also proves that the pyruvate metabolism is re-directed from organic acid fermentation to ethanol production. Further work has to be performed to fully understand pdc and adhB optimal working conditions and to optimize the environment for a high yield ethanol production.
 <div align="right">
@@ Line 319: / Line 337: @@
 <tr align="center">
   <td>     0</td><td>     36 </td>
-  <td>  ~0 </td>
+  <td>   0.05 [~0 ; 0.15] </td>
-  <td>     59 </td> <td>  ~0</td>
+  <td>     59 </td> <td>   0.03 [  0.02 ;   0.04]</td>
   </tr><tr align="center">
-  <td>    25</td><td>     37 </td>
+  <td>    25</td><td>     32 </td>
-  <td>  ~0 </td>
+  <td>   0.12 [~0 ; 0.19] </td>
-  <td>     65 </td> <td>   0.03 [ ~0 ;   0.19]</td>
+  <td>     61 </td> <td>   0.03 [  0.02 ;   0.04]</td>
   </tr><tr align="center">
-  <td>    50</td><td>     40 </td>
+  <td>    50</td><td>     34 </td>
-  <td>   0.86 [  0.82 ;   0.92] </td>
+  <td>   0.17 [0.05; 0.25] </td>
-  <td>     65 </td> <td>   1.19 [  0.53 ;   3.08]</td>
+  <td>     57 </td> <td>   0.04 [  0.03 ;   0.05]</td>
   </tr><tr align="center">
   <td>    75</td><td>     43 </td>
-  <td>   4.44 [  3.81 ;   5.44] </td>
+  <td>   0.15 [~0; 0.23] </td>
-  <td>     73 </td> <td>   4.29 [  2.63 ;   7.83]</td>
+  <td>     65 </td> <td>   0.03 [  0.02 ;   0.06]</td>
   </tr><tr align="center">
-  <td>   100</td><td>     43 </td>
+  <td>   100</td><td>     40 </td>
-  <td>   4.53 [  3.88 ;   5.18] </td>
+  <td>   0.21 [0.19; 0.23] </td>
-  <td>     76 </td> <td>   5.23 [  3.21 ;   9.28]</td>
+  <td>     62 </td> <td>   0.03 [  0.03 ;   0.05]</td>
   </tr><tr align="center">
-  <td>   200</td><td>     39 </td>
+  <td>   200</td><td>     40 </td>
-  <td>   3.53 [  3.35 ;   3.75] </td>
+  <td>   0.25 [0.20; 0.28] </td>
-  <td>     73 </td> <td>   4.99 [  3.14 ;   8.45]</td>
+  <td>     70 </td> <td>   0.04 [  0.03 ;   0.06]</td>
   </tr><tr align="center">
-  <td>   300</td><td>     41 </td>
+  <td>   300</td><td>     43 </td>
-  <td>   4.04 [  3.56 ;   4.57] </td>
+  <td>   0.20 [0.18; 0.21] </td>
-  <td>     62 </td> <td>   5.40 [  3.39 ;   9.56]</td>
+  <td>     67 </td> <td>   0.05 [  0.04 ;   0.08]</td>
   </tr>
 </table>
@@ Line 355: / Line 373: @@
 |[[Image:PV_A9_LB.png|700px|thumb|<partinfo>BBa_K173009</partinfo> Growth curves for BBa_K173009 in LB]]
 |-
-|[[Image:PV_A9_Scell_LB.png|700px|thumb|<partinfo>BBa_K173009</partinfo>(dGFP/dt)/OD in LB and M9]]
+|[[Image:PV_A9_Scell_LB.png|700px|thumb|<partinfo>BBa_K173009</partinfo>(dGFP/dt)/OD in LB]]
 |-
-|[[Image:PV_A9_Scell_M9.png|700px|thumb|<partinfo>BBa_K173009</partinfo>(dGFP/dt)/OD in LB and M9]]
+|[[Image:PV_A9_Scell_M9.png|700px|thumb|<partinfo>BBa_K173009</partinfo>(dGFP/dt)/OD in M9]]
 |-
 |[[Image:PV_A9_IndCurvM9.png|700px|thumb|<partinfo>BBa_K173009</partinfo> Induction curve of BBa_K173009 in M9]]
@@ Line 367: / Line 385: @@
 We demonstrated that this part works as expected, sensing the aTc concentration provided in the culture medium. The transfer function of this sensor has been characterized in standard units (RPUs) in two different growth media (LB and M9 supplemented with glycerol), as well as the metabolic burden (in terms of doubling time) which affects E. coli bearing this part.
-On the other hand, we did not expect to have a higher GFP synthesis rate per cell after the exponential growth phase than in the exponential phase itself (as reported in the 3rd plot).
+On the other hand, we did not expect to have a higher GFP synthesis rate per cell after the exponential growth phase than in the exponential phase itself (as reported in the 3rd plot for both LB and M9).
-This parts shows to have a very low leakage rate (about 0.025 RPU) but also a very low induction for high concentrations of aTc. So it can be used for tight regulation, in those systems who need very low leakage rates (no gene expressed in absence of inductor) and a response not important.
+This part shows to have a very low leakage rate (about 0.025 RPU) but also a very low transcription rate for high concentrations of aTc. So, it can be used for tight regulation, in those systems who need very low leakage rates (no gene expressed in absence of inducer) but not for massive protein production.
+Unfortunately, the estimated transfer function is very noisy (probably because the measured fluorescence values are very close to blank values), and further experiments should be performed in order to validate these induction curves.
 <div align="right">
@@ Line 407: / Line 427: @@
   <td>      0 </td>
 <td>     36 </td>
-  <td>   0.51 [  0.43 ;   0.73] </td>
+  <td>   0.51 [0.43; 0.73] </td>
-  <td>     78 </td> <td>   0.96 [  0.80 ;   1.23]</td>
+  <td>     78 </td> <td>   0.96 [0.80; 1.23]</td>
 </tr><tr align="center">
   <td>     25 </td>
 <td>     33 </td>
-  <td>   1.36 [  1.28 ;   1.44] </td>
+  <td>   1.36 [1.28; 1.44] </td>
-  <td>     82 </td> <td>   1.66 [  1.38 ;   2.11]</td>
+  <td>     82 </td> <td>   1.66 [1.38; 2.11]</td>
 </tr><tr align="center">
   <td>     50 </td>
 <td>     40 </td>
-  <td>   1.39 [  1.30 ;   1.48] </td>
+  <td>   1.39 [1.30; 1.48] </td>
-  <td>     81 </td> <td>   1.85 [  1.61 ;   2.27]</td>
+  <td>     81 </td> <td>   1.85 [1.61; 2.27]</td>
 </tr><tr align="center">
   <td>     75 </td>
 <td>     43 </td>
-  <td>   1.25 [  1.17 ;   1.31] </td>
+  <td>   1.25 [1.17; 1.31] </td>
-  <td>     88 </td> <td>   1.75 [  1.51 ;   2.15]</td>
+  <td>     88 </td> <td>   1.75 [1.51; 2.15]</td>
 </tr><tr align="center">
   <td>    100 </td>
 <td>     43 </td>
-  <td>   1.40 [  1.28 ;   1.48] </td>
+  <td>   1.40 [1.28; 1.48] </td>
-  <td>     90 </td> <td>   1.89 [  1.68 ;   2.34]</td>
+  <td>     90 </td> <td>   1.89 [1.68; 2.34]</td>
 </tr><tr align="center">
@@ Line 438: / Line 458: @@
 <td>     45 </td>
   <td>   1.37 [  1.31 ;   1.41] </td>
-  <td>     92 </td> <td>   1.75 [  1.51 ;   2.09]</td>
+  <td>     92 </td> <td>   1.75 [1.51; 2.09]</td>
 </tr><tr align="center">
@@ Line 444: / Line 464: @@
 <td>     48 </td>
   <td>   1.45 [  1.36 ;   1.55] </td>
-  <td>     94 </td> <td>   1.73 [  1.53 ;   2.07]</td>
+  <td>     94 </td> <td>   1.73 [1.53; 2.07]</td>
 </tr>
@@ Line 470: / Line 490: @@
 On the other hand, as for BBa_K173007, we did not expect to have a higher GFP synthesis rate per cell after the exponential growth phase than in the exponential phase itself (as reported in the 3rd plot).
-This part shows a high level of leakage, of about 1.5 RPU but also high levels of induction for high concentrations of aTc. For this reason, it can be used in those cases where an important response in term of gene expression is required in presence of inductor, but is not suitabel if the absence of expression has to be absten when inductor is absent.
+This part shows a high level of leakage (about 0.5 RPU in LB and 1 RPU in M9) but also high levels of induction for high concentrations of aTc (about 1.45 RPU in LB and 1.7 RPU in M9). For this reason, it can be used in those cases where an important response in terms of gene expression is required in presence of an inducer, but is not suitabe if the absence of expression has to be off when inducer is absent. Its induction range is 0.95 RPU in LB and 0.7 RPU in M9.
+The switch point of this device is between 0 ng/ml and 25 ng/ml of aTc in both LB and M9.
 <div align="right">
@@ Line 489: / Line 511: @@
 ===Characterization===
+*We inoculated 8 ul of <partinfo>BBa_K173012</partinfo> glycerol stock in 5 ml of LB + Amp and incubated it overnight (37°C, 220 rpm).
+*In the morning we diluted 1:100 the culture in two falcon tubes and incubated them under the same conditions as before.
+*After about 6 hours we added IPTG in one of the two cultures (final concentration = 1 mM).
+*Fluorescence and O.D.600 were measured in the microplate reader after additional 4 hours.
+Results: both cultures had a comparable O.D.600, but also had the same RFU values, demonstrating that the induced culture did not produce any GFP.
 ===Conclusions===