Team:UNIPV-Pavia/Project/PromotoriAuto

From 2010.igem.org




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: BBa_K300009 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 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
BBa_J23100 2547
BBa_J231011791
BBa_J23105623
BBa_J231061185
BBa_J23110844
BBa_J23114256
BBa_J23116396
BBa_J231181429

Before constructing the signal generators, BBa_K300009 and BBa_K300010 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. 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 (BBa_J61002)
Figure 6 - R.P.U. of the studied promoters from Anderson promoters' collection, M9 medium and high copy plasmid (BBa_J61002)
Figure 7 - R.P.U. of the studied promoters from Anderson promoters' collection, M9 medium and low copy plasmid (pSB4C5). These plasmids were constructed by assembling the EcoRI-PstI fragment of BBa_J61002-BBa_J231xx in pSB4C5 vector, in order to transfer the promoter and the RBS-RFP-TT expression construct from BBa_J61002 to pSB4C5.

Doubling times were evaluated for the described cultures (HC stands for High Copy and LC stands for Low Copy):

Promoter doubling time [minutes]
LB in HC plasmid M9 in HC plasmid M9 in LC plasmid
BBa_J23100 33.75
±
1.34
82.53
±
2.45
86.11
±
4.45
BBa_J23101 35.93
±
0.62
82.68
±
1.84
86.42
±
1.91
BBa_J23105 29.86
±
0.33
63.09
±
7.08
85.00
±
5.13
BBa_J23106 29.17
±
0.96
68.11
±
4.25
88.71
±
0.90
BBa_J23110 31.28
±
0.42
67.52
±
5.87
76.15
±
2.16
BBa_J23114 28.97
±
0.49
59.44
±
5.20
80.12
±
0.95
BBa_J23116 28.14
±
0.25
72.74
±
0.37
81.68
±
3.08
BBa_J23118 32.84
±
0.31
73.64
±
2.41
89.86
±
2.93

Discussion: we observed that the ranking previously documented in the Registry is not valid in all the tested conditions, even if a general agreement can be observed. As an example, BBa_J23110 in high copy plasmid is stronger than BBa_J23118, 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: BBa_K300009 and BBa_K300010 were assembled downstream of the above mentioned promoters, thus obtaining the following parts:

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

Some of the promoters could not be cloned upstream of these devices because they produced amounts of LuxI protein that give a high metabolic burden for E. coli, so it was not possible to study all the combinations as transformants could not be obtained in some cases. For each part, a measurement system was built, exploiting the production of the reporter gene GFP (Green Fluorescent 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 by autoinducer generators

Experimental implementation: The autoinducer generators BBa_K300030, BBa_K300028, BBa_K300029, BBa_K300025, BBa_K300026 and BBa_K300027 were, thus, characterized by measuring the concentration of HSL released in the medium of cultures grown for 6 hours. All the details are available in this section.

BBa_T9002 contained in pSB1A3 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 in high copy plasmid pSB1A2 is reported in Fig.8 and in the table below:

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

The amount of 3OC6-HSL produced by the parts contained in low copy plasmid pSB4C5 after a 6 hour cell growth is reported in Fig.9 and in the table below:

Figure 9 - BBa_T9002 calibration curve for detection of [HSL] produced by the autoinducer generators in low copy vector.


BioBrick Wiki name E. coli strain [HSL]
BBa_K300030 I14 DH5alpha 0.005 uM
BBa_K300028 I15 DH5alpha 0.002 uM
BBa_K300029 I16 DH5alpha not detected
BBa_K300025 I17 DH5alpha 0.003 uM
BBa_K300026 I18 DH5alpha not detected
BBa_K300027 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 BBa_K300017, BBa_K300014, BBa_K300015, BBa_K300016 and BBa_K300012) in high copy number plasmid pSB1A2 or low copy number plasmid pSB4C5. A third alternative was the assembly of signal generator on a low copy number plasmid (pSB4C5) and the receiver device on high copy number plasmid (pSB1A2). The circuits we obtained and tested are summarized in tables.


Sender/Receiver devices assembled as a unique BioBrick part on the same vector
BioBrick
Sender
Description Sender/Receiver Device Vector BioBrick composite part
BBa_K300030 Pv SignalGeneratorDevice.png
J23118
pSB1A2
HC
BBa_K300017
BBa_K300028 Pv SignalGeneratorDevice.png
J23110
pSB1A2
HC
BBa_K300014
BBa_K300029 Pv SignalGeneratorDevice.png
J23116
pSB1A2
HC
BBa_K300016
BBa_K300026 Pv SignalGeneratorDevice.png
J23105
pSB1A2
HC
BBa_K300012
xxx Pv SignalGeneratorDevice.png
J23114
pSB1A2
HC
BBa_K300015
BBa_K300030 Pv SignalGeneratorDevice.png
J23118
pSB4C5
LC
BBa_K300017
BBa_K300028 Pv SignalGeneratorDevice.png
J23110
pSB4C5
LC
BBa_K300014
BBa_K300029 Pv SignalGeneratorDevice.png
J23116
pSB4C5
LC
BBa_K300016
BBa_K300026 Pv SignalGeneratorDevice.png
J23105
pSB4C5
LC
BBa_K300012
Sender devices assembled on low copy number vector and Receiver device on high copy number vector
BioBrick
Sender
Description Sender Vector BBa_F2620
Receiver vector
BBa_K300030 Pv SignalGeneratorDevice.png
J23118
pSB4C5
LC
pSB1A2
HC
BBa_K300028 Pv SignalGeneratorDevice.png
J23110
pSB4C5
LC
pSB1A2
HC
BBa_K300029 Pv SignalGeneratorDevice.png
J23116
pSB4C5
LC
pSB1A2
HC
BBa_K300025 Pv SignalGeneratorDevice.png
J23101
pSB4C5
LC
pSB1A2
HC
BBa_K300026 Pv SignalGeneratorDevice.png
J23105
pSB4C5
LC
pSB1A2
HC
BBa_K300027 Pv SignalGeneratorDevice.png
J23106
pSB4C5
LC
pSB1A2
HC

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
BBa_K300030 in pSB1A2
Pv SignalGeneratorDevice.png
J23118
BBa_T9002 in pSB1A2
Pv T9002.png
BBa_K300024
in pSB1A2
BBa_K300028 in pSB1A2
Pv SignalGeneratorDevice.png
J23110
BBa_T9002 in pSB1A2
Pv T9002.png
BBa_K300021
in pSB1A2
BBa_K300029 in pSB1A2
Pv SignalGeneratorDevice.png
J23116
BBa_T9002 in pSB1A2
Pv T9002.png
BBa_K300022
in pSB1A2
BBa_K300026 in pSB1A2
Pv SignalGeneratorDevice.png
J23105
BBa_T9002 in pSB1A2
Pv T9002.png
BBa_K300019
in pSB1A2
xxx
Pv SignalGeneratorDevice.png
J23114
BBa_T9002 in pSB1A2
Pv T9002.png
BBa_K300023
in pSB1A2
BBa_K300030 in pSB4C5
Pv SignalGeneratorDevice.png
J23118
BBa_T9002 in pSB4C5
Pv T9002.png
BBa_K300024
in pSB4C5
BBa_K300028 in pSB4C5
Pv SignalGeneratorDevice.png
J23110
BBa_T9002 in pSB4C5
Pv T9002.png
BBa_K300021
in pSB4C5
BBa_K300029 in pSB4C5
Pv SignalGeneratorDevice.png
J23116
BBa_T9002 in pSB4C5
Pv T9002.png
BBa_K300022
in pSB4C5
BBa_K300026 in pSB4C5
Pv SignalGeneratorDevice.png
J23105
BBa_T9002 in pSB4C5
Pv T9002.png
BBa_K300019
in pSB4C5
BBa_K300030 in pSB4C5
Pv SignalGeneratorDevice.png
J23118
BBa_T9002 in pSB1A3
Pv T9002.png
Sender and Receiver are contained
in two different plasmids,
cotransformed in the same cell
BBa_K300028 in pSB4C5
Pv SignalGeneratorDevice.png
J23110
BBa_T9002 in pSB1A3
Pv T9002.png
Sender and Receiver are contained
in two different plasmids,
cotransformed in the same cell
BBa_K300029 in pSB4C5
Pv SignalGeneratorDevice.png
J23116
BBa_T9002 in pSB1A3
Pv T9002.png
Sender and Receiver are contained
in two different plasmids,
cotransformed in the same cell
BBa_K300026 in pSB4C5
Pv SignalGeneratorDevice.png
J23105
BBa_T9002 in pSB1A3
Pv T9002.png
Sender and Receiver are contained
in two different plasmids,
cotransformed in the same cell
BBa_K300025 in pSB4C5
Pv SignalGeneratorDevice.png
J23101
BBa_T9002 in pSB1A3
Pv T9002.png
Sender and Receiver are contained
in two different plasmids,
cotransformed in the same cell
BBa_K300027 in pSB4C5
Pv SignalGeneratorDevice.png
J23106
BBa_T9002 in pSB1A3
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 GFP were grown according to this protocol and all data collected were analyzed as explained in this section. An example of O.D.600 and fluorescence signals for a self-inducible device expressing GFP (BBa_K300026), as well as its Scell signal and the estimated threshold value, is reported below.

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

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 synthesis 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 BBa_J23101 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 pSB1A2

Self-inducible device
working
not working
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
BBa_K300017 (wiki name: I7) in pSB1A2 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
BBa_K300014 (wiki name: I8) in pSB1A2 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 **
BBa_K300015 (wiki name: I9) in pSB1A2 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
BBa_K300016 (wiki name: I10) in pSB1A2 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
BBa_K300012 (wiki name: I12) in pSB1A2 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 pSB4C5

Self-inducible device
working
not working
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
BBa_K300017 (wiki name: I7) in pSB4C5 plasmid
Pv BBa K300017.png
X X X X 0.24
±
0.0004 **
not computed 62.41
±
1.56 **
not computed
BBa_K300014 (wiki name: I8) in pSB4C5 plasmid


Pv BBa K300014.png

X X X X X X X X
BBa_K300015 (wiki name: I9) in pSB4C5 plasmid


Pv BBa K300015.png

X X X X X X X X
BBa_K300016 (wiki name: I10) in pSB4C5 plasmid


Pv BBa K300016.png

X X X X X X X X
BBa_K300012 (wiki name: I12) in pSB4C5 plasmid


Pv BBa K300012.png

X X X X X X X X


Tab. 3 - Sender on low copy plasmid pSB4C5 and Receiver on high copy plasmid pSB1A3

Self-inducible device
working
not working
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
BBa_K300030
(wiki name: I14)
in pSB4C5 plasmid

BBa_T9002 in pSB1A3
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
BBa_K300028
(wiki name: I15)
in pSB4C5 plasmid

BBa_T9002 in pSB1A3
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
BBa_K300029 (wiki name: I16) in pSB4C5 plasmid BBa_T9002 in pSB1A3
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
BBa_K300025
(wiki name: I17)
in pSB4C5 plasmid
BBa_T9002 in pSB1A3
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
BBa_K300026
(wiki name: I18)
in pSB4C5 plasmid
BBa_T9002 in pSB1A3
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 **
BBa_K300027
(wiki name: I19)
in pSB4C5 plasmid
BBa_T9002 in pSB1A3
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 algorithm. 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 on two independent experiments.

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

Discussion: two modular PoPS-based devices (BBa_K300010 and BBa_K300009/BBa_F2620) were designed and used to realize a library of self-inducible devices, able to start the production of the heterologous protein at a defined culture density. They were 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 graphical summary is reported in the figures below:

Typical E. coli growth curve with O.D.start evaluated by Threshold algorithm in LB
Typical E. coli growth curve with O.D.start 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 the figures below, 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 for BBa_K300010 (Sender&Receiver device in HC plasmid) and for BBa_K300009/BBa_F2620 (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 BBa_K300010 in high copy number plasmid in M9 medium
O.D.start and K_HSL as a function of RPUs of the promoters controlling the autoinducer production for BBa_K300010 in low copy number plasmid used in combination with BBa_F2620 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 the reported graphs. This is consistent with the expected behaviour of these parts, since the HSL synthesis rate is an increasing function of the upstream promoter's strength.

For RPU values greater or equal to 1, the expected behaviour is not confirmed anymore. This is probably because too high luxI expression levels and/or synthesis rate of HSL are injurious for the cell.

The combination of promoter strength variation and plasmid copy number modulation allows the creation of a library of self-inducible devices able to perform autoinduction at many different O.D.600 values, in any cellular growth phase.