Team:UNIPV-Pavia/Project/PromotoriAuto

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Revision as of 13:48, 27 October 2010




ProteInProgress: a cellular assembly line for protein manufacturing



Motivation Solutions
Implementation & Results

References


Implementation and Results: Self-Inducible promoters




Self-inducible promoters


Integrative standard vector for E. coli


Integrative standard vector for yeast


Purification of proteins

Self-inducible promoters

Regulation of signal protein production

Experimental implementation: <partinfo>BBa_K300009</partinfo> part was assembled downstream of different constitutive promoters, thus obtaining a signal molecule generator. The choice of constitutive promoters was performed between the ones belonging to the [http://partsregistry.org/Part:BBa_J23101 Anderson’s promoters collection]; we chose promoters according to their activities reported in the Registry of Standard Biological Parts, in order to have a thick mesh:

Promoter Strength (a.u.)
reported in the Registry
<partinfo>BBa_J23100</partinfo> 2547
<partinfo>BBa_J23101</partinfo>1791
<partinfo>BBa_J23105</partinfo>623
<partinfo>BBa_J23106</partinfo>1185
<partinfo>BBa_J23110</partinfo>844
<partinfo>BBa_J23114</partinfo>256
<partinfo>BBa_J23116</partinfo>396
<partinfo>BBa_J23118</partinfo>1429

Before constructing the signal generators, <partinfo>BBa_K300009</partinfo> and <partinfo>BBa_K300010</partinfo> under the regulation of one of these constitutive promoters, we evaluated the promoter activities in Relative Promoter Units (R.P.U.) according to Data analysis for RPU evaluation, using the reporter protein RFP (Red Fluorescent Protein) in different experimental conditions (plasmids’ copy number and growth medium), many of them not yet explored and documented:

  • high copy number plasmids and LB;
  • high copy number plasmids and M9;
  • low copy number plasmids and M9.

It was not possible to evaluate promoters activities in low copy number plasmids in LB because the RFP activity was too weak and not distinguishable from the background. RFP fluorescence and Optical Density at 600nm (O.D.600) were measured in 96-well microplates, as reported in Microplate reader experiments for constitutive promoters (R.P.U. evaluation) - Protocol #2 and data were analyzed as reported in Data analysis for RPU evaluation;

Results: results are shown here.

Figure 5 - R.P.U. of the studied promoters from Anderson promoters' collection, LB medium and high copy plasmid (<partinfo>BBa_J61002</partinfo>)
Figure 6 - R.P.U. of the studied promoters from Anderson promoters' collection, M9 medium and high copy plasmid (<partinfo>BBa_J61002</partinfo>)
Figure 7 - R.P.U. of the studied promoters from Anderson promoters' collection, M9 medium and low copy plasmid (<partinfo>pSB4C5</partinfo>). These plasmids were constructed by assembling the EcoRI-PstI the <partinfo>BBa_J61002</partinfo>-BBa_J231xx EcoRI-PstI fragment in <partinfo>pSB4C5</partinfo>, in order to transfer the RBS-RFP-TT expression construct from <partinfo>BBa_J61002</partinfo> to <partinfo>pSB4C5</partinfo>.

Doubling times were evaluated for the described cultures:

Promoter doubling time [minutes]
LB in HC plasmid M9 in HC plasmid M9 in LC plasmid
<partinfo>BBa_J23100</partinfo>
<partinfo>BBa_J23101</partinfo>
<partinfo>BBa_J23105</partinfo>
<partinfo>BBa_J23106</partinfo>
<partinfo>BBa_J23110</partinfo>
<partinfo>BBa_J23114</partinfo>
<partinfo>BBa_J23116</partinfo>
<partinfo>BBa_J23118</partinfo>

Discussion: we observed that the ranking previously documented in the Registry is not valid in all the conditions, even if a general agreement can be observed. As an example, <partinfo>BBa_J23110</partinfo> in high copy plasmid is stronger than <partinfo>BBa_J23118</partinfo>, in contrast with the ranking reported in the Registry.


After the evaluation of promoter activity, signal generators were constructed in high copy and low copy plasmids: <partinfo>BBa_K300009</partinfo> and <partinfo>BBa_K300010</partinfo> were assembled downstream of the above mentioned promoters, thus obtaining the following parts:

BioBrick Description
<partinfo>BBa_K300030</partinfo> Pv SignalGeneratorDevice.png
J23118
<partinfo>BBa_K300028</partinfo> Pv SignalGeneratorDevice.png
J23110
<partinfo>BBa_K300029</partinfo> Pv SignalGeneratorDevice.png
J23116
<partinfo>BBa_K300025</partinfo> Pv SignalGeneratorDevice.png
J23101
<partinfo>BBa_K300026</partinfo> Pv SignalGeneratorDevice.png
J23105
<partinfo>BBa_K300027</partinfo> Pv SignalGeneratorDevice.png
J23106
<partinfo>BBa_K300017</partinfo> Pv SignalGeneratorSensorDevice.png
J23118
<partinfo>BBa_K300014</partinfo> Pv SignalGeneratorSensorDevice.png
J23110
<partinfo>BBa_K300015</partinfo> Pv SignalGeneratorSensorDevice.png
J23114
<partinfo>BBa_K300016</partinfo> Pv SignalGeneratorSensorDevice.png
J23116
<partinfo>BBa_K300012</partinfo> Pv SignalGeneratorSensorDevice.png
J23105

Some of the promoters could not be cloned upstream of these devices because they produced LuxI protein amounts that give a high metabolic burden for E. coli, so it was not possible to study all the combinations as transformans could not be obtained in some cases. For each part, a measurement system was built, exploiting the production of the reporter gene GFP (Green Fluoresent Protein) to evaluate the "switch on" condition of every self-inducible promoter. Many different combinations were explored, in order to provide a library of promoters able to initiate transcription at the desired culture density.

Quantification of the HSL produced

Experimental implementation The new parts were, thus, characterized, measuring the HSL concentration released in the medium after a 6 hour growth of the cultures. All the details are available in this section.

<partinfo>BBa_T9002</partinfo> contained in <partinfo>pSB1A3</partinfo> in E. coli TOP10 was used as a HSL->GFP biosensor. In every experiment, a HSL-GFP calibration curve with known concentration of HSL was produced.

Results The amount of 3OC6-HSL produced after a 6 hours growth by E. coli DH5alpha bearing the parts contained in high copy plasmid <partinfo>pSB1A2</partinfo> is reported in Fig.8 and in the table:

Figure 8 - <partinfo>BBa_T9002</partinfo> calibration curve for detection of [HSL] produced in high copy plasmid
BioBrick Wiki name E. coli strain [HSL]
<partinfo>BBa_K300030</partinfo> I14 DH5alpha 0.7 uM
<partinfo>BBa_K300028</partinfo> I15 DH5alpha 0.04 uM
<partinfo>BBa_K300029</partinfo> I16 DH5alpha not detected
<partinfo>BBa_K300025</partinfo> I17 DH5alpha 0.09 uM
<partinfo>BBa_K300026</partinfo> I18 DH5alpha not detected
<partinfo>BBa_K300027</partinfo> I19 DH5alpha 0.002 uM

The amount of 3OC6-HSL produced after 6 hours growth by the parts contained in low copy plasmid <partinfo>pSB4C5</partinfo> is reported in Fig.9 and in the table:

Figure 9 - <partinfo>BBa_T9002</partinfo> calibration curve for detection of [HSL] produced in low copy plasmid


BioBrick Wiki name E. coli strain [HSL]
<partinfo>BBa_K300030</partinfo> I14 DH5alpha 0.005 uM
<partinfo>BBa_K300028</partinfo> I15 DH5alpha 0.002 uM
<partinfo>BBa_K300029</partinfo> I16 DH5alpha not detected
<partinfo>BBa_K300025</partinfo> I17 DH5alpha 0.003 uM
<partinfo>BBa_K300026</partinfo> I18 DH5alpha not detected
<partinfo>BBa_K300027</partinfo> I19 DH5alpha not detected

Discussion These experiments provided extremely useful informations about the capability of the signal generators to produce the 3OC6-HSL signal molecule. Data are quantitative, but incomplete because for weak promoters or medium-strength promoters contained in a low copy number plasmid the amount of 3OC6-HSL was not detectable using this system. However, this simple experiment shows that there is a strong correlation between the strength of promoter and the amount of signal molecule produced. These results confirm that the production of the autoinducer can be engineered in E. coli and different expression systems reach different amounts of 3OC6-HSL in the growth media as a function of the promoter strength. Thus, these results demonstrate that self-inducible circuits can be rationally designed from a set of well characterized standard parts.

Modulation of plasmid copy number

Signal generator and sensor device were assembled in an unique part (such as <partinfo>BBa_K300017</partinfo>, <partinfo>BBa_K300014</partinfo>, <partinfo>BBa_K300015</partinfo>, <partinfo>BBa_K300016</partinfo> and <partinfo>BBa_K300012</partinfo>) beared on high copy number plasmid <partinfo>pSB1A2</partinfo> or low copy number plasmid <partinfo>pSb4C5</partinfo>. A third alternative was the assembly of signal generator on a low copy number plasmid (<partinfo>pSB4C5</partinfo>) and the receiver device on high copy number plasmid (<partinfo>pSB1A2</partinfo>). The circuits we obtained and tested are summarized here:


BioBrick
Sender
Description Sender Vector <partinfo>BBa_F2620</partinfo>
Receiver vector
BioBrick composite part
<partinfo>BBa_K300030</partinfo> Pv SignalGeneratorDevice.png
J23118
<partinfo>pSB1A2</partinfo>
HC
<partinfo>BBa_K300017</partinfo>
<partinfo>BBa_K300028</partinfo> Pv SignalGeneratorDevice.png
J23110
<partinfo>pSB1A2</partinfo>
HC
<partinfo>BBa_K300014</partinfo>
<partinfo>BBa_K300029</partinfo> Pv SignalGeneratorDevice.png
J23116
<partinfo>pSB1A2</partinfo>
HC
<partinfo>BBa_K300016</partinfo>
<partinfo>BBa_K300026</partinfo> Pv SignalGeneratorDevice.png
J23105
<partinfo>pSB1A2</partinfo>
HC
<partinfo>BBa_K300012</partinfo>
xxx Pv SignalGeneratorDevice.png
J23114
<partinfo>pSB1A2</partinfo>
HC
<partinfo>BBa_K300015</partinfo>
<partinfo>BBa_K300030</partinfo> Pv SignalGeneratorDevice.png
J23118
<partinfo>pSB4C5</partinfo>
LC
<partinfo>BBa_K300017</partinfo>
<partinfo>BBa_K300028</partinfo> Pv SignalGeneratorDevice.png
J23110
<partinfo>pSB4C5</partinfo>
LC
<partinfo>BBa_K300014</partinfo>
<partinfo>BBa_K300029</partinfo> Pv SignalGeneratorDevice.png
J23116
<partinfo>pSB4C5</partinfo>
LC
<partinfo>BBa_K300016</partinfo>
<partinfo>BBa_K300026</partinfo> Pv SignalGeneratorDevice.png
J23105
<partinfo>pSB4C5</partinfo>
LC
<partinfo>BBa_K300012</partinfo>
<partinfo>BBa_K300030</partinfo> Pv SignalGeneratorDevice.png
J23118
<partinfo>pSB4C5</partinfo>
LC
<partinfo>pSB1A2</partinfo>
HC
Parts are contained in two different vectors
<partinfo>BBa_K300028</partinfo> Pv SignalGeneratorDevice.png
J23110
<partinfo>pSB4C5</partinfo>
LC
<partinfo>pSB1A2</partinfo>
HC
Parts are contained in two different vectors
<partinfo>BBa_K300029</partinfo> Pv SignalGeneratorDevice.png
J23116
<partinfo>pSB4C5</partinfo>
LC
<partinfo>pSB1A2</partinfo>
HC
Parts are contained in two different vectors
<partinfo>BBa_K300025</partinfo> Pv SignalGeneratorDevice.png
J23101
<partinfo>pSB4C5</partinfo>
LC
<partinfo>pSB1A2</partinfo>
HC
Parts are contained in two different vectors
<partinfo>BBa_K300026</partinfo> Pv SignalGeneratorDevice.png
J23105
<partinfo>pSB4C5</partinfo>
LC
<partinfo>pSB1A2</partinfo>
HC
Parts are contained in two different vectors
<partinfo>BBa_K300027</partinfo> Pv SignalGeneratorDevice.png
J23106
<partinfo>pSB4C5</partinfo>
LC
<partinfo>pSB1A2</partinfo>
HC
Parts are contained in two different vectors

Results

The following measurement systems were realized assembling GFP downstream of each self-inducible device. The parts characterized are reported in this table:

Sender device Sensor systems with GFP Measurement Device
<partinfo>BBa_K300030</partinfo> in <partinfo>pSB1A2</partinfo>
Pv SignalGeneratorDevice.png
J23118
<partinfo>BBa_T9002</partinfo> in <partinfo>pSB1A2</partinfo>
Pv T9002.png
<partinfo>BBa_K300024</partinfo>
in <partinfo>pSB1A2</partinfo>
<partinfo>BBa_K300028</partinfo> in <partinfo>pSB1A2</partinfo>
Pv SignalGeneratorDevice.png
J23110
<partinfo>BBa_T9002</partinfo> in <partinfo>pSB1A2</partinfo>
Pv T9002.png
<partinfo>BBa_K300021</partinfo>
in <partinfo>pSB1A2</partinfo>
<partinfo>BBa_K300029</partinfo> in <partinfo>pSB1A2</partinfo>
Pv SignalGeneratorDevice.png
J23116
<partinfo>BBa_T9002</partinfo> in <partinfo>pSB1A2</partinfo>
Pv T9002.png
<partinfo>BBa_K300022</partinfo>
in <partinfo>pSB1A2</partinfo>
<partinfo>BBa_K300026</partinfo> in <partinfo>pSB1A2</partinfo>
Pv SignalGeneratorDevice.png
J23105
<partinfo>BBa_T9002</partinfo> in <partinfo>pSB1A2</partinfo>
Pv T9002.png
<partinfo>BBa_K300019</partinfo>
in <partinfo>pSB1A2</partinfo>
xxx
Pv SignalGeneratorDevice.png
J23114
<partinfo>BBa_T9002</partinfo> in <partinfo>pSB1A2</partinfo>
Pv T9002.png
<partinfo>BBa_K300023</partinfo>
in <partinfo>pSB1A2</partinfo>
<partinfo>BBa_K300030</partinfo> in <partinfo>pSB4C5</partinfo>
Pv SignalGeneratorDevice.png
J23118
<partinfo>BBa_T9002</partinfo> in <partinfo>pSB4C5</partinfo>
Pv T9002.png
<partinfo>BBa_K300024</partinfo>
in <partinfo>pSB4C5</partinfo>
<partinfo>BBa_K300028</partinfo> in <partinfo>pSB4C5</partinfo>
Pv SignalGeneratorDevice.png
J23110
<partinfo>BBa_T9002</partinfo> in <partinfo>pSB4C5</partinfo>
Pv T9002.png
<partinfo>BBa_K300021</partinfo>
in <partinfo>pSB4C5</partinfo>
<partinfo>BBa_K300029</partinfo> in <partinfo>pSB4C5</partinfo>
Pv SignalGeneratorDevice.png
J23116
<partinfo>BBa_T9002</partinfo> in <partinfo>pSB4C5</partinfo>
Pv T9002.png
<partinfo>BBa_K300022</partinfo>
in <partinfo>pSB4C5</partinfo>
<partinfo>BBa_K300026</partinfo> in <partinfo>pSB4C5</partinfo>
Pv SignalGeneratorDevice.png
J23105
<partinfo>BBa_T9002</partinfo> in <partinfo>pSB4C5</partinfo>
Pv T9002.png
<partinfo>BBa_K300019</partinfo>
in <partinfo>pSB4C5</partinfo>
<partinfo>BBa_K300030</partinfo> in <partinfo>pSB4C5</partinfo>
Pv SignalGeneratorDevice.png
J23118
<partinfo>BBa_T9002</partinfo> in <partinfo>pSB1A3</partinfo>
Pv T9002.png
Sender and Receiver are contained in two different plasmids,
cotransformed in the same cell
<partinfo>BBa_K300028</partinfo> in <partinfo>pSB4C5</partinfo>
Pv SignalGeneratorDevice.png
J23110
<partinfo>BBa_T9002</partinfo> in <partinfo>pSB1A3</partinfo>
Pv T9002.png
Sender and Receiver are contained in two different plasmids,
cotransformed in the same cell
<partinfo>BBa_K300029</partinfo> in <partinfo>pSB4C5</partinfo>
Pv SignalGeneratorDevice.png
J23116
<partinfo>BBa_T9002</partinfo> in <partinfo>pSB1A3</partinfo>
Pv T9002.png
Sender and Receiver are contained
in two different plasmids,
cotransformed in the same cell
<partinfo>BBa_K300026</partinfo> in <partinfo>pSB4C5</partinfo>
Pv SignalGeneratorDevice.png
J23105
<partinfo>BBa_T9002</partinfo> in <partinfo>pSB1A3</partinfo>
Pv T9002.png
Sender and Receiver are contained
in two different plasmids,
cotransformed in the same cell
<partinfo>BBa_K300025</partinfo> in <partinfo>pSB4C5</partinfo>
Pv SignalGeneratorDevice.png
J23101
<partinfo>BBa_T9002</partinfo> in <partinfo>pSB1A3</partinfo>
Pv T9002.png
Sender and Receiver are contained
in two different plasmids,
cotransformed in the same cell
<partinfo>BBa_K300027</partinfo> in <partinfo>pSB4C5</partinfo>
Pv SignalGeneratorDevice.png
J23106
<partinfo>BBa_T9002</partinfo> in <partinfo>pSB1A3</partinfo>
Pv T9002.png
Sender and Receiver are contained
in two different plasmids,
cotransformed in the same cell

Cultures of E. coli TOP10 bearing the plasmids containing the self-inducible devices expressing G.F.P. were grown according to this protocol and all data collected were analyzed as explained in this section

Growth curve of <partinfo>BBa_K300019</partinfo> (O.D.600)
Fluorescence curve of <partinfo>BBa_K300019</partinfo> (G.F.P.)
Fluorescence VS Optical density curve of <partinfo>BBa_K300019</partinfo>
Scell=(dGFP/dt)/O.D.600 and threshold

Thus, these BioBrick parts can be used to express recombinant proteins without adding an inducer to trigger the transcription of their genes; in large-scale production of such proteins this strategy could be also cost saving.

For every self-inducible device, several parameters were evaluated:

  • O.D.start is the O.D.600 corresponding to the transcription initiation of the gene of interest, it was evaluated as reported in this section
  • K_HSL is the HSL synthesys rate per cell, it was estimated with the algorithm described here
  • Doubling time is the period of time required for a cell population to double. It was evaluated as described in Doubling time evaluation section
  • Scell_ratio was evaluated as (Scell_max_Phi)/(Scell_max_J101). Phi is the self-inducible device of ineterst, J101 is the reference standard <partinfo>BBa_J23101</partinfo> contained in the same vector of the receiver device. Scell_max_phi was evaluated for times subsequent to the transcription initiation.

Results are summarized in the following tables:


Tab. 1 - Sender and Receiver on high copy plasmid <partinfo>pSB1A2</partinfo>

Self-inducible device
working
not working
Description LB M9
O.D.start K_HSL
[nmol/min]
Doubling time
[min]
Scell ratio O.D.start K_HSL
[nmol/min]
Doubling time
[min]
Scell ratio
<partinfo>BBa_K300017</partinfo> (wiki name: I7) in <partinfo>pSB1A2</partinfo> plasmid Pv BBa K300017.png Constitutive 3.11 10^-16
±
2.23 10^-17
31.15
±
2.52
0.95
±
0.15
0.027
±
0.002
1.52 10^-15
±
9.76 10^-17
61.28
±
2.67
1.68
±
0.23
<partinfo>BBa_K300014</partinfo> (wiki name: I8) in <partinfo>pSB1A2</partinfo> plasmid Pv BBa K300014.png X X 27.86
±
1.14
X 0.13
±
0.023 **
3.59 10^-16
±
1.22 10^-16 **
0.37
±
0.13 **
53.09
±
1.18 **
<partinfo>BBa_K300015</partinfo> (wiki name: I9) in <partinfo>pSB1A2</partinfo> plasmid Pv BBa K300015.png 0.33
±
*
8.94 10^-17
±
*
33.81
±
3.03
0.98
±
*
0.38
±
0.02
3.78 10^-17
±
4.65 10^-18
57.20
±
1.32
0.07
±
0.01
<partinfo>BBa_K300016</partinfo> (wiki name: I10) in <partinfo>pSB1A2</partinfo> plasmid Pv BBa K300016.png 0.54
±
*
7.53 10^-18
±
*
39.55
±
0.32
0.21
±
*
0.32
±
0.02
5.70 10^-17
±
7.75 10^-18
55.33
±
5.19
0.13
±
0.02
<partinfo>BBa_K300012</partinfo> (wiki name: I12) in <partinfo>pSB1A2</partinfo> plasmid Pv BBa K300012.png 0.50
±
0.01
1.16 10^-17
±
6.41 10^-19
36.66
±
2.50
0.58
±
0.02
0.30
±
0.03
6.53 10^-17
±
1.43 10^-17
50.92
±
2.92
0.21
±
0.06

Tab. 2 - Sender and Receiver on low copy plasmid <partinfo>pSB4C5</partinfo>

Self-inducible device
working
not working
Description LB M9
O.D.start K_HSL
[nmol/min]
Doubling time
[min]
Scell ratio O.D.start K_HSL
[nmol/min]
Doubling time
[min]
Scell ratio
<partinfo>BBa_K300017</partinfo> (wiki name: I7) in <partinfo>pSB4C5</partinfo> plasmid Pv BBa K300017.png X X X X 0.24
±
0.0004 **
not computed 62.41
±
1.56 **
not computed
<partinfo>BBa_K300014</partinfo> (wiki name: I8) in <partinfo>pSB4C5</partinfo> plasmid Pv BBa K300014.png X X X X X X X X
<partinfo>BBa_K300015</partinfo> (wiki name: I9) in <partinfo>pSB4C5</partinfo> plasmid Pv BBa K300015.png X X X X X X X X
<partinfo>BBa_K300016</partinfo> (wiki name: I10) in <partinfo>pSB4C5</partinfo> plasmid Pv BBa K300016.png X X X X X X X X
<partinfo>BBa_K300012</partinfo> (wiki name: I12) in <partinfo>pSB4C5</partinfo> plasmid Pv BBa K300012.png X X X X X X X X

Tab. 3 - Sender on low copy plasmid <partinfo>pSB4C5</partinfo> and Receiver on high copy plasmid <partinfo>pSB1A3</partinfo>

Self-inducible device
working
not working
Description LB M9
O.D.start K_HSL
[nmol/min]
Doubling time
[min]
Scell ratio O.D.start K_HSL
[nmol/min]
Doubling time
[min]
Scell ratio
<partinfo>BBa_K300030</partinfo>
(wiki name: I14)
in <partinfo>pSB4C5</partinfo> plasmid
Pv BBa K300030.png
LC
Pv BBa F2620.png
HC
Constitutive 2.92 10^-16
±
8.16 10^-18
33.20
±
1.46
1.20
±
0.20
0.05
±
0.005
8.06 10^-16
±
1.28 10^-16
58.27
±
5.66
0.91
±
0.15
<partinfo>BBa_K300028</partinfo>
(wiki name: I15)
in <partinfo>pSB4C5</partinfo> plasmid
Pv BBa K300028.png
LC
Pv BBa F2620.png
HC
0.32
±
0.04 **
8.31 10^-17
±
3.92 10^-17 **
33.24
±
1.27
0.61
±
0.04 **
0.15
±
0.007
1.72 10^-16
±
6.65 10^-18
65.57
±
5.99
0.26
±
0.03
<partinfo>BBa_K300029</partinfo>
(wiki name: I16)
in <partinfo>pSB4C5</partinfo> plasmid
Pv BBa K300029.png
LC
Pv BBa F2620.png
HC
0.31
±
*
1.17 10^-16
±
*
35.46
±
2.85
0.57
±
*
X X X X
<partinfo>BBa_K300025</partinfo>
(wiki name: I17)
in <partinfo>pSB4C5</partinfo> plasmid
Pv BBa K300025.png
LC
Pv BBa F2620.png
HC
Constitutive 2.85 10^-16
±
1.90 10^-17
33.27
±
2.85
1.09
±
0.28
0.1
±
0.01
3.81 10^-16
±
4.68 10^-17
59.02
±
8.28
0.45
±
0.08
<partinfo>BBa_K300026</partinfo>
(wiki name: I18)
in <partinfo>pSB4C5</partinfo> plasmid
Pv BBa K300026.png
LC
Pv BBa F2620.png
HC
X X 34.63
±
1.31
X 0.53
±
0.03 **
1.78 10^-17
±
1.36 10^-18 **
61.68
±
7.08
0.03
±
0.002 **
<partinfo>BBa_K300027</partinfo>
(wiki name: I19)
in <partinfo>pSB4C5</partinfo> plasmid
Pv BBa K300027.png
LC
Pv BBa F2620.png
HC
X X 33.80
±
1.78
X 0.33
±
0.05
5.46 10^-17
±
7.86 10^-18
52.84
±
4.29
0.06
±
0.002

LEGEND OF TABLES:

Constitutive: the induction point, in term of O.D.600, is under the minimum detectable value calculated by the aglorithm. This minimum value was estimated by running the algorithm on data acquired from a culture that constitutively produces GFP. For this reason, the devices labelled as constitutive can be considered as constitutive GFP producers.

*: in two of three experiments the self-induction failed, thus having a non-induced culture for all the cell densities. The standard errors were not computed for these cultures.

**: in one of three experiments the self-induction failed, thus having a non-induced culture for all the cell densities. The standard errors were computed computed on two independent experiments.

<partinfo>BBa_K300016</partinfo> is labelled with *, but probably induction failed in two of the three experiments because the culture didn't reach the ODstart point (the experiment was stopped before the culture reached the O.D.600 critical value).

Discussion: two self-inducible devices (<partinfo>BBa_K300010</partinfo> and <partinfo>BBa_K300009</partinfo>/<partinfo>BBa_F2620</partinfo>) were realized and characterized in many different experimental conditions:

  • varying the strength of the promoter controlling the production of the signal molecule (Sender Modulation)
  • varying the copy number of vectors bearing both Sender and Receiver circuits
  • varying the growth medium (LB or M9)

A library of self-inducible promoters, able to start the production of the heterologous protein at a defined culture density, was realized. A graphical summary is reported in Figures:

Average growth curve with ODstart evaluated by Threshold algorithm in LB
Average growth curve with ODstart evaluated by Threshold algorithm in M9

A model-based approach was proposed to estimate many interesting parameters, such as the HSL synthesis rate per cell and an algorithm was proposed, in order to evaluate the O.D.start for every self-inducible device.

In figure, the O.D.start and the HSL synthesis rate as a function of the strength of the promoter (RPU) controlling the signal molecule production are reported, both for <partinfo>BBa_K300010</partinfo> (Sender&Receiver device in HC plasmid) and for <partinfo>BBa_K300009</partinfo>/<partinfo>BBa_F2620</partinfo> (Sender device in LC plasmid in combination with Receiver device in HC plasmid).

O.D.start and K_HSL as a function of RPUs of the promoters controlling the signal molecule production for <partinfo>BBa_K300010</partinfo> in high copy number plasmid in M9 medium
O.D.start and K_HSL as a function of RPUs of the promoters controlling the Signal Molucule production for <partinfo>BBa_K300010</partinfo> in low copy number plasmid used in combination with <partinfo>BBa_F2620</partinfo> in high copy plasmid in M9 medium

A strong correlation between the promoter strength, previously measured in RPU, and the O.D.start is depicted by data. This is consistent with the expected behaviour of these parts, since the HSL synthesis rate results stronger at the increase of promoter's strength.

For RPU values greater or equal to 1, the expected behaviour is not confirmed anymore. This is probably due to the fact that too high synthesis rate of HSL are injurious for the cell. A range of RPU values for promoters that control the Sender device was identified. When the PoPs-in signal belongs to this range, the expected behaviour is confirmed by experimental data. The combination of promoter strength variation and plasmid copy number modulation allows the creation of a library of self-inducible devices, able to start the production of a target protein at different O.D._start values, in any cellular growth phase.