Team:UNIPV-Pavia/New Parts

From 2009.igem.org

(Difference between revisions)
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__NOTOC__
__NOTOC__
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<html>
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<!--Da qui si mettono le new parts -->
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</html>
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= '''Our new parts''' =
==BBa_K173003 - ethanol producing device==
==BBa_K173003 - ethanol producing device==
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</div>
</div>
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==BBa_K173007 - aTc inducible device with J23100 promoter==
 +
===Description===
 +
This is an aTc sensing device.
 +
 +
<partinfo>BBa_J23100</partinfo> promoter drives the constitutive production of tetR repressor (<partinfo>BBa_C0040</partinfo>), which inhibits tetR promoter (<partinfo>BBa_R0040</partinfo>) 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.
 +
 +
[[Image:Pv_A6_BBa_K173007.png]]
 +
 +
The data below are referred to <partinfo>BBa_K173009</partinfo>, which is the measurement system of <partinfo>BBa_K173007</partinfo>.
 +
 +
===Characterization===
 +
 +
<html>
 +
<table border=1 align="center" cellpadding=5px>
 +
 +
<tr>
 +
<td rowspan=2 valign="bottom" align="center"><b>aTc concentration<br>[ng/ml]</b></td>
 +
<td colspan=2 align="center"><b>LB</b></td>
 +
<td colspan=2 align="center"><b>M9 supplemented</b></td>
 +
</tr>
 +
 +
<tr>
 +
 +
<td><b>Doubling time [minutes]</b></td>
 +
<td><b>RPU</b></td>
 +
<td><b>Doubling time [minutes]</b></td>
 +
<td><b>RPU</b></td>
 +
</tr>
 +
<tr align="center">
 +
<td>0</td><td> 35.560000 </td>
 +
<td> 0.048800 [-0.113900 ; 0.148700] </td>
 +
<td> 59.270000 </td> <td> 0.026600 [0.020900 ; 0.036400]</td>
 +
</tr><tr align="center">
 +
<td>25</td><td> 32.230000 </td>
 +
<td> 0.123100 [-0.017400 ; 0.192300] </td>
 +
<td> 61.430000 </td> <td> 0.032800 [0.021600 ; 0.044000]</td>
 +
</tr><tr align="center">
 +
<td>50</td><td> 34.500000 </td>
 +
<td> 0.173600 [0.052200 ; 0.254000] </td>
 +
<td> 56.740000 </td> <td> 0.037100 [0.029500 ; 0.046500]</td>
 +
</tr><tr align="center">
 +
<td>75</td><td> 43.110000 </td>
 +
<td> 0.146200 [-0.059200 ; 0.234600] </td>
 +
<td> 65.010000 </td> <td> 0.033400 [0.020600 ; 0.058100]</td>
 +
</tr><tr align="center">
 +
<td>100</td><td> 40.400000 </td>
 +
<td> 0.210800 [0.188500 ; 0.228200] </td>
 +
<td> 61.590000 </td> <td> 0.032900 [0.027000 ; 0.047100]</td>
 +
</tr><tr align="center">
 +
<td>200</td><td> 40.200000 </td>
 +
<td> 0.247400 [0.195400 ; 0.279700] </td>
 +
<td> 69.730000 </td> <td> 0.039300 [0.031000 ; 0.061400]</td>
 +
</tr><tr align="center">
 +
<td>300</td><td> 43.420000 </td>
 +
<td> 0.198200 [0.182800 ; 0.208300] </td>
 +
<td> 66.610000 </td> <td> 0.050900 [0.037200 ; 0.075100]</td>
 +
</tr>
 +
</table>
 +
</html>
 +
 +
 +
{|align="center"
 +
|[[Image:PV_A9_M9.png|700px|thumb|<partinfo>BBa_K173009</partinfo> Growth curves for BBa_K173009 in M9]]
 +
|-
 +
|[[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_M9.png|700px|thumb|<partinfo>BBa_K173009</partinfo>(dGFP/dt)/OD in LB and M9]]
 +
|-
 +
|[[Image:PV_A9_IndCurvM9.png|700px|thumb|<partinfo>BBa_K173009</partinfo> Induction curve of BBa_K173009 in M9]]
 +
|-
 +
|[[Image:PV_A9_IndCurvLB.png|700px|thumb|<partinfo>BBa_K173009</partinfo> 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).
 +
 +
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.
 +
 +
<div align="right">
 +
[[#top|Top]]
 +
</div>
 +
 +
==BBa_K173011 - aTc inducible device with J23118 promoter==
 +
===Description===
 +
This is an aTc sensing device.
 +
 +
<partinfo>BBa_J23118</partinfo> promoter drives the constitutive production of tetR repressor (<partinfo>BBa_C0040</partinfo>), which inhibits tetR promoter (<partinfo>BBa_R0040</partinfo>) 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 <partinfo>BBa_K173007</partinfo> 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 <partinfo>BBa_K173026</partinfo>, which is the measurement system of <partinfo>BBa_K173011</partinfo>.
 +
 +
[[Image:pv_A12_description.png]]
 +
 +
===Characterization===
 +
<html>
 +
<table border=1 align="center" cellpadding=5px>
 +
 +
<tr>
 +
<td rowspan=2 valign="bottom" align="center"><b>aTc concentration<br>[ng/ml]</b></td>
 +
<td colspan=2 align="center"><b>LB</b></td>
 +
<td colspan=2 align="center"><b>M9 supplemented</b></td>
 +
</tr>
 +
 +
<tr>
 +
 +
<td><b>Doubling time [minutes]</b></td>
 +
<td><b>RPU</b></td>
 +
<td><b>Doubling time [minutes]</b></td>
 +
<td><b>RPU</b></td>
 +
</tr>
 +
<tr align="center">
 +
<td> 0 </td>
 +
<td> 35.510000 </td>
 +
<td> 0.513000 [0.429600 ; 0.728000] </td>
 +
<td> 77.860000 </td> <td> 0.962500 [0.800400 ; 1.226600]</td>
 +
 
 +
</tr><tr align="center">
 +
<td> 25 </td>
 +
<td> 33.350000 </td>
 +
<td> 1.363900 [1.278700 ; 1.442400] </td>
 +
<td> 82.200000 </td> <td> 1.658800 [1.384200 ; 2.111400]</td>
 +
 
 +
</tr><tr align="center">
 +
<td> 50 </td>
 +
<td> 40.320000 </td>
 +
<td> 1.389500 [1.304300 ; 1.481900] </td>
 +
<td> 80.700000 </td> <td> 1.850400 [1.609100 ; 2.267100]</td>
 +
 
 +
</tr><tr align="center">
 +
<td> 75 </td>
 +
<td> 42.620000 </td>
 +
<td> 1.255000 [1.171400 ; 1.313200] </td>
 +
<td> 88.080000 </td> <td> 1.748200 [1.509600 ; 2.145700]</td>
 +
 
 +
</tr><tr align="center">
 +
<td> 100 </td>
 +
<td> 43.440000 </td>
 +
<td> 1.399800 [1.279500 ; 1.475600] </td>
 +
<td> 90.280000 </td> <td> 1.885300 [1.679900 ; 2.337500]</td>
 +
 
 +
</tr><tr align="center">
 +
<td> 200 </td>
 +
<td> 45.070000 </td>
 +
<td> 1.370900 [1.308800 ; 1.412900] </td>
 +
<td> 92.270000 </td> <td> 1.745000 [1.514800 ; 2.087300]</td>
 +
 
 +
</tr><tr align="center">
 +
<td> 300 </td>
 +
<td> 48.060000 </td>
 +
<td> 1.448600 [1.355900 ; 1.551000] </td>
 +
<td> 93.870000 </td> <td> 1.728900 [1.534000 ; 2.071100]</td>
 +
 
 +
</tr>
 +
 +
</table>
 +
</html>
 +
 +
{|align="center"
 +
|[[Image:PV_A16_M9.png|700px|thumb|<partinfo>BBa_K173026</partinfo> Growth curves for BBa_K173026 in M9]]
 +
|-
 +
|[[Image:PV_A16_LB.png|700px|thumb|<partinfo>BBa_K173026</partinfo> Growth curves for BBa_K173026 in LB]]
 +
|-
 +
|[[Image:PV_A16_Scell_LB.png|700px|thumb|<partinfo>BBa_K173026</partinfo>(dGFP/dt)/OD in LB and M9]]
 +
|-
 +
|[[Image:PV_A16_Scell_M9.png|700px|thumb|<partinfo>BBa_K173026</partinfo>(dGFP/dt)/OD in LB and M9]]
 +
|-
 +
|[[Image:PV_A16_IndCurvM9.png|700px|thumb|<partinfo>BBa_K173026</partinfo> Induction curve of BBa_K173026 in M9]]
 +
|-
 +
|[[Image:PV_A16_IndCurvLB.png|700px|thumb|<partinfo>BBa_K173026</partinfo> 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, 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.
 +
 +
<div align="right">
 +
[[#top|Top]]
 +
</div>
 +
 +
==BBa_K173010 - lactose/IPTG inducible device with J23118 promoter==
 +
===Description===
 +
This should work as a lactose/IPTG sensor.
 +
 +
<partinfo>BBa_J23118</partinfo> promoter drives the constitutive production of lacI repressor (<partinfo>BBa_C0012</partinfo>), which inhibits lac promoter (<partinfo>BBa_R0011</partinfo>) 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 (<partinfo>BBa_R0011</partinfo>), designed taking the Plambda promoter (<partinfo>BBa_R0051</partinfo>) and substituting its cI (<partinfo>BBa_C0051</partinfo>) 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 <partinfo>BBa_K173012</partinfo>, which is the measurement system of <partinfo>BBa_K173010</partinfo> itself.
 +
 +
[[Image:pv_A11_description.png]]
 +
 +
===Characterization===
 +
 +
===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 <partinfo>BBa_B0033</partinfo> negative control.
 +
 +
Unfortunately, we did not check if the sequence of <partinfo>BBa_K173012</partinfo>, 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.
 +
<div align="right">
 +
[[#top|Top]]
 +
</div>
 +
 +
<html>
 +
<!--Fine new parts -->
 +
</html>
</td>
</td>
</tr>
</tr>
</table>
</table>

Revision as of 10:34, 21 October 2009

EthanolPVanimation.gif



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


Our new parts

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 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.

Pv A5new2 3 BBa K173003.png

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 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):

promoter upstream of plasmid promoter RPU in LB colony phenotype
? small colonies
not estimated small colonies
? small colonies
? without induction normal size colonies
not estimated normal size 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. 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.


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 centrifugationcentrifugation
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 high induction (1 uM). 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

pH

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:

PROTOCOL#1: pH at the end of fermentation (after the SECOND INOCULUM).

PROTOCOL#2: pH after the FIRST and the SECOND (end of fermentation) INOCULUM

PROTOCOL#3: pH after the FIRST and the SECOND (end of fermentation) INOCULUM

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 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.

Growth curves 

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  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.

Growth curves of , in pSB1AK3, in pSB4C5 + 1uM HSL, in pSB1A2 negative control, in pSB1AK3 negative control

This test showed that in pSB1AK3 is able to reach much higher OD600 than the other cultures, reaching ? values. in pSB4C5 both induced and uninduced had comparable growth curves and reached OD600 of about ?. Negative controls ( in pSB1A2 and in pSB1AK3) had comparable growth curves and reached low OD (about ?). It is surprising that 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 in pSB1AK3 and in pSB4C5 produce less organic acids than the negative controls, hopefully thanks to pdc and adhB and consequently grow better.


Ethanol production Here we report the fermentation results of 100 g/l of glucose:

PROTOCOL#1: ethanol at the end of fermentation.

PROTOCOL#2: ethanol at the end of fermentation.

PROTOCOL#2: ethanol at the end of fermentation.

PROTOCOL#3: ethanol at the end of fermentation.

Conclusions

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.

Pv A6 BBa K173007.png

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

Characterization

aTc concentration
[ng/ml]		 LB M9 supplemented
Doubling time [minutes] RPU Doubling time [minutes] RPU
0 35.560000 0.048800 [-0.113900 ; 0.148700] 59.270000 0.026600 [0.020900 ; 0.036400]
25 32.230000 0.123100 [-0.017400 ; 0.192300] 61.430000 0.032800 [0.021600 ; 0.044000]
50 34.500000 0.173600 [0.052200 ; 0.254000] 56.740000 0.037100 [0.029500 ; 0.046500]
75 43.110000 0.146200 [-0.059200 ; 0.234600] 65.010000 0.033400 [0.020600 ; 0.058100]
100 40.400000 0.210800 [0.188500 ; 0.228200] 61.590000 0.032900 [0.027000 ; 0.047100]
200 40.200000 0.247400 [0.195400 ; 0.279700] 69.730000 0.039300 [0.031000 ; 0.061400]
300 43.420000 0.198200 [0.182800 ; 0.208300] 66.610000 0.050900 [0.037200 ; 0.075100]


Growth curves for BBa_K173009 in M9
Growth curves for BBa_K173009 in LB
(dGFP/dt)/OD in LB and M9
(dGFP/dt)/OD in LB and 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).

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.

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 .

Pv A12 description.png

Characterization

aTc concentration
[ng/ml]		 LB M9 supplemented
Doubling time [minutes] RPU Doubling time [minutes] RPU
0 35.510000 0.513000 [0.429600 ; 0.728000] 77.860000 0.962500 [0.800400 ; 1.226600]
25 33.350000 1.363900 [1.278700 ; 1.442400] 82.200000 1.658800 [1.384200 ; 2.111400]
50 40.320000 1.389500 [1.304300 ; 1.481900] 80.700000 1.850400 [1.609100 ; 2.267100]
75 42.620000 1.255000 [1.171400 ; 1.313200] 88.080000 1.748200 [1.509600 ; 2.145700]
100 43.440000 1.399800 [1.279500 ; 1.475600] 90.280000 1.885300 [1.679900 ; 2.337500]
200 45.070000 1.370900 [1.308800 ; 1.412900] 92.270000 1.745000 [1.514800 ; 2.087300]
300 48.060000 1.448600 [1.355900 ; 1.551000] 93.870000 1.728900 [1.534000 ; 2.071100]

Growth curves for BBa_K173026 in M9
Growth curves for BBa_K173026 in LB
(dGFP/dt)/OD in LB and M9
(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, 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.

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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.

Pv A11 description.png

Characterization

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.

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