ProteInProgress: a cellular assembly line for protein manufacturing

Implementation and Results: Self-Inducible promoters

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:

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 and 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 time were evaluated for the described cultures:

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>	J23118
<partinfo>BBa_K300028</partinfo>	J23110
<partinfo>BBa_K300029</partinfo>	J23116
<partinfo>BBa_K300025</partinfo>	J23101
<partinfo>BBa_K300026</partinfo>	J23105
<partinfo>BBa_K300027</partinfo>	J23106
<partinfo>BBa_K300017</partinfo>	J23118
<partinfo>BBa_K300014</partinfo>	J23110
<partinfo>BBa_K300015</partinfo>	J23114
<partinfo>BBa_K300016</partinfo>	J23116
<partinfo>BBa_K300012</partinfo>	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

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

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>	J23118	<partinfo>pSB1A2</partinfo> HC		<partinfo>BBa_K300017</partinfo>
<partinfo>BBa_K300028</partinfo>	J23110	<partinfo>pSB1A2</partinfo> HC		<partinfo>BBa_K300014</partinfo>
<partinfo>BBa_K300029</partinfo>	J23116	<partinfo>pSB1A2</partinfo> HC		<partinfo>BBa_K300016</partinfo>
<partinfo>BBa_K300026</partinfo>	J23105	<partinfo>pSB1A2</partinfo> HC		<partinfo>BBa_K300012</partinfo>
xxx	J23114	<partinfo>pSB1A2</partinfo> HC		<partinfo>BBa_K300015</partinfo>
<partinfo>BBa_K300030</partinfo>	J23118	<partinfo>pSB4C5</partinfo> LC		<partinfo>BBa_K300017</partinfo>
<partinfo>BBa_K300028</partinfo>	J23110	<partinfo>pSB4C5</partinfo> LC		<partinfo>BBa_K300014</partinfo>
<partinfo>BBa_K300029</partinfo>	J23116	<partinfo>pSB4C5</partinfo> LC		<partinfo>BBa_K300016</partinfo>
<partinfo>BBa_K300026</partinfo>	J23105	<partinfo>pSB4C5</partinfo> LC		<partinfo>BBa_K300012</partinfo>
<partinfo>BBa_K300030</partinfo>	J23118	<partinfo>pSB4C5</partinfo> LC	<partinfo>pSB1A2</partinfo> HC	Parts are contained in two different vectors
<partinfo>BBa_K300028</partinfo>	J23110	<partinfo>pSB4C5</partinfo> LC	<partinfo>pSB1A2</partinfo> HC	Parts are contained in two different vectors
<partinfo>BBa_K300029</partinfo>	J23116	<partinfo>pSB4C5</partinfo> LC	<partinfo>pSB1A2</partinfo> HC	Parts are contained in two different vectors
<partinfo>BBa_K300025</partinfo>	J23101	<partinfo>pSB4C5</partinfo> LC	<partinfo>pSB1A2</partinfo> HC	Parts are contained in two different vectors
<partinfo>BBa_K300026</partinfo>	J23105	<partinfo>pSB4C5</partinfo> LC	<partinfo>pSB1A2</partinfo> HC	Parts are contained in two different vectors
<partinfo>BBa_K300027</partinfo>	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> J23118	<partinfo>BBa_T9002</partinfo> in <partinfo>pSB1A2</partinfo>	<partinfo>BBa_K300024</partinfo> in <partinfo>pSB1A2</partinfo>
<partinfo>BBa_K300028</partinfo> in <partinfo>pSB1A2</partinfo> J23110	<partinfo>BBa_T9002</partinfo> in <partinfo>pSB1A2</partinfo>	<partinfo>BBa_K300021</partinfo> in <partinfo>pSB1A2</partinfo>
<partinfo>BBa_K300029</partinfo> in <partinfo>pSB1A2</partinfo> J23116	<partinfo>BBa_T9002</partinfo> in <partinfo>pSB1A2</partinfo>	<partinfo>BBa_K300022</partinfo> in <partinfo>pSB1A2</partinfo>
<partinfo>BBa_K300026</partinfo> in <partinfo>pSB1A2</partinfo> J23105	<partinfo>BBa_T9002</partinfo> in <partinfo>pSB1A2</partinfo>	<partinfo>BBa_K300019</partinfo> in <partinfo>pSB1A2</partinfo>
xxx J23114	<partinfo>BBa_T9002</partinfo> in <partinfo>pSB1A2</partinfo>	<partinfo>BBa_K300023</partinfo> in <partinfo>pSB1A2</partinfo>
<partinfo>BBa_K300030</partinfo> in <partinfo>pSB4C5</partinfo> J23118	<partinfo>BBa_T9002</partinfo> in <partinfo>pSB4C5</partinfo>	<partinfo>BBa_K300024</partinfo> in <partinfo>pSB4C5</partinfo>
<partinfo>BBa_K300028</partinfo> in <partinfo>pSB4C5</partinfo> J23110	<partinfo>BBa_T9002</partinfo> in <partinfo>pSB4C5</partinfo>	<partinfo>BBa_K300021</partinfo> in <partinfo>pSB4C5</partinfo>
<partinfo>BBa_K300029</partinfo> in <partinfo>pSB4C5</partinfo> J23116	<partinfo>BBa_T9002</partinfo> in <partinfo>pSB4C5</partinfo>	<partinfo>BBa_K300022</partinfo> in <partinfo>pSB4C5</partinfo>
<partinfo>BBa_K300026</partinfo> in <partinfo>pSB4C5</partinfo> J23105	<partinfo>BBa_T9002</partinfo> in <partinfo>pSB4C5</partinfo>	<partinfo>BBa_K300019</partinfo> in <partinfo>pSB4C5</partinfo>
<partinfo>BBa_K300030</partinfo> in <partinfo>pSB4C5</partinfo> J23118	<partinfo>BBa_T9002</partinfo> in <partinfo>pSB1A3</partinfo>	Sender and Receiver are contained in two different plasmids, cotransformed in the same cell
<partinfo>BBa_K300028</partinfo> in <partinfo>pSB4C5</partinfo> J23110	<partinfo>BBa_T9002</partinfo> in <partinfo>pSB1A3</partinfo>	Sender and Receiver are contained in two different plasmids, cotransformed in the same cell
<partinfo>BBa_K300029</partinfo> in <partinfo>pSB4C5</partinfo> J23116	<partinfo>BBa_T9002</partinfo> in <partinfo>pSB1A3</partinfo>	Sender and Receiver are contained in two different plasmids, cotransformed in the same cell
<partinfo>BBa_K300026</partinfo> in <partinfo>pSB4C5</partinfo> J23105	<partinfo>BBa_T9002</partinfo> in <partinfo>pSB1A3</partinfo>	Sender and Receiver are contained in two different plasmids, cotransformed in the same cell
<partinfo>BBa_K300025</partinfo> in <partinfo>pSB4C5</partinfo> J23101	<partinfo>BBa_T9002</partinfo> in <partinfo>pSB1A3</partinfo>	Sender and Receiver are contained in two different plasmids, cotransformed in the same cell
<partinfo>BBa_K300027</partinfo> in <partinfo>pSB4C5</partinfo> J23106	<partinfo>BBa_T9002</partinfo> in <partinfo>pSB1A3</partinfo>	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 ineterest, 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	Doubling time	Scell ratio	O.D.start	K_HSL	Doubling time	Scell ratio
<partinfo>BBa_K300017</partinfo> (wiki name: I7) in <partinfo>pSB1A2</partinfo> plasmid		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		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		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		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		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	Doubling time	Scell ratio	O.D.start	K_HSL	Doubling time	Scell ratio
<partinfo>BBa_K300017</partinfo> (wiki name: I7) in <partinfo>pSB4C5</partinfo> plasmid		X	X	X	X	0.24 ± 0.0004 **	not computed	62.41 ± 1.56 **	0.08 ± 0.008 **
<partinfo>BBa_K300014</partinfo> (wiki name: I8) in <partinfo>pSB4C5</partinfo> plasmid		X	X	X	X	X	X	X	X
<partinfo>BBa_K300015</partinfo> (wiki name: I9) in <partinfo>pSB4C5</partinfo> plasmid		X	X	X	X	X	X	X	X
<partinfo>BBa_K300016</partinfo> (wiki name: I10) in <partinfo>pSB4C5</partinfo> plasmid		X	X	X	X	X	X	X	X
<partinfo>BBa_K300012</partinfo> (wiki name: I12) in <partinfo>pSB4C5</partinfo> plasmid		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	Doubling time	Scell ratio	O.D.start	K_HSL	Doubling time	Scell ratio
<partinfo>BBa_K300030</partinfo> (wiki name: I14) in <partinfo>pSB4C5</partinfo> plasmid	LC	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	LC	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	LC	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	LC	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	LC	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	LC	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

Discussion: a self-inducer device was realized and char

Average growth curve with ODstart evaluated by Threshold algorithm in LB

Average growth curve with ODstart evaluated by Threshold algorithm in M9