Experiments

Introduction

iGEM Sheffield Biological Context

Growth modulation is a foundational-level biotechnology, with multiple applications. PARSE aims to produce a proof of concept plasmid that enables the modulation of the growth of E. coli in a fine-tunable manner, in this case, using an IPTG-inducible promoter (pLac) and phage-derived growth slowing (GS) genes as modulators. Additionally, it aims to lay a foundation whereby any user can calibrate their own inducible systems and utilise their own reporters to achieve a similar objective.

To achieve this, the devised strategy was to assemble the following constructs:

1. Anderson promoter- vsfGFP-0 in pET28a: A fluorescent reporter expressed by a range of constitutive promoters of known strength relative to the wild type (J23119).
2. pLac- vsfGFP-0 in pET28a: A newly characterised fluorescent reporter expressed by an inducible promoter.
3. pLac- GS in pET28a : A GS expressed by an inducible promoter.

pET28a was the vector of choice due to its inbuilt Lac inducible system, most importantly containing pLac, an IPTG-inducible promoter (further considerations are explained here ). As a result, a wide range of levels of gene expression can be obtained by varying the concentrations of IPTG.

The purpose behind the first set of plasmids is to establish a baseline for gene expression measurements in terms of a constant scale. The second plasmids allow calibration of any inducible system (Lac in this case) in relation to the Anderson scale. The third set of plasmids, allow to test the functionality of the GS as well as to characterise them thoroughly by providing a link between the level of gene expression and the bacterial growth achieved.

The four Anderson promoters used in assembly were J23100, J23106, J23110 and J23114, from strongest to weakest promoter strength respectively (Figure 1), and the six GS that were successfully inserted into pET28a are listed in Figure 2.

The purpose of computational modelling is to determine the strength of the inducible promoters (used in plasmid constructs 2 and 3) that express the green fluorescent protein gene, causing the transformed bacteria to fluoresce. The strength of each promoter is quantified by the amount of fluorescence per cell to describe the amount of gene expression of the reporter gene within the microbial culture. Additionally, the levels of the gene expression of the GS could be further visualised with the predicted decrease of the specific growth rate. See our biomodelling page for more information.

Hypothesis

Growth rate is a crucial parameter to be considered during the expression of genetic constructs in bacteria as it gives insight into the relation between gene product generation and the metabolic state of the culture [1]. Gene expression and biomass increase remain constant as long as nutrient requirements are satisfied. Otherwise, output efficiency is reduced [2,3]. The development of a strategy to actively regulate the growth rate will ensure a constant product output and will open the possibility of including intermediate growth rates when convenient.

Growth modulator genes can be used as synthetic biology tools to achieve this objective. Their effect on bacterial growth can be positive (“growth enhancer”) or negative (“growth slower”). The latter includes several phage-derived genes which hinder host DNA and/or mRNA synthesis and thus growth rate while the expression of phage-derived genes is maintained [4]. The assembly of an artificial genetic system that takes advantage of growth-slower genes to precisely control bacterial growth requires tight control of their expression in the first place. Our proposed system is composed of a plasmid containing one growth-slower gene expressed under an IPTG-inducible Lac promoter. A calibration with plasmids containing Anderson promoters and GFP-containing plasmids relates the presence of a reporter molecule to the expression level of our genetic unit and makes it possible to link this overall expression with the observed values of optical density (OD) in the culture.

Our working hypothesis is that the expression of our growth slower-based genetic system will result in a decrease in the bacterial growth rate depending on the promoter strength. The activation of the system (with an inducer molecule such as IPTG) will be reflected in a lower growth rate of the culture. The growth inhibition level will be proportional to the concentration of the inductor molecule.

Anderson calibration

At the first stage of the project, four plasmids that combined a promoter from the collection of Anderson promoters of different strengths and one fluorescent reporter gene (vsfGFP-0) were constructed in a pET28a vector (Figure 1) and transformed into E. coli BL21-Lemo bacteria.

Through using these plasmids, promoters of specific strengths (Table 1) (which have been extensively characterised and quantified) can be linked to the respective level of gene expression they produce, using the fluorescence of the reporter gene as a measure of it. This makes these plasmids essential as a first step towards the calibration of a fine-tunable inducible system.

Promoter	Measured Strength
J23100	1.00
J23106	0.47
J23110	0.33
J23114	0.10

Fluorescence was measured at EM: 535nm and, EX: 485nm (these were the closest wavelengths available in the plate reader used. The protein properties are actually EM: 510nm and EX: 485nm). The expected standard curve that can be plotted from these results is shown in Figure 4, where higher measured fluorescence values correspond to Anderson promoters of higher strengths. Optical Density was also measured so that a normalised value representing “fluorescence per cell” could be extracted.

The second set of plasmids constructed involves utilising the Lac IPTG-inducible promoter the pET28a contains by default, and coupling it with the same reporter gene (vsfGFP-0) to measure the different levels of fluorescence generated by the constructs when induced with different concentrations of IPTG [5]. (Figure 5). This allows to relate the constant Anderson promoter strengths to the concentrations of inducer, a more arbitrary parameter, and build a standard curve in relation to inducer concentration, which will make the desired system more fine-tuneable than the discrete and limited catalogue of Anderson promoters.

The expected standard curve is shown in Figure 6, where increasing concentrations of inducer result in higher levels of fluorescence. Results of the experiments conducted can be consulted here.

It is important to stress that, anyone who intends to use PARSE may choose their own reporter gene as well as their own inducible system. The calibration of their inducible system should be done in a similar fashion, using the Anderson promoters as the link between a consistent measuring tool and an inducible mechanism to be calibrated.

The last combination of plasmids built contain pLac coupled to one of six growth slowing genes (see Table 2 and Figure 7- initially we planned on testing seven but did not manage to clone gp0.7). Inducing the transformed cells with IPTG should slow down their growth as the growth slowing genes begin to be expressed. Their growth repressor function mechanism has been previously described in literature (See References)

Gene	Target in Host	Reference
gp79	RNAP (core)	[ 6 ]
gp104	Dnal (helicase loader)	[ 7 ]
gp2	RNAP (β′ subunit)	[ 8 ]
gp240	DNA Pol III (β subunit)	[ 9 ]
asiA	σ70 factor	[ 10 ]
alc	RNAP (core)	[ 11 ]

Experiments to measure optical density of E. coli transformed with these plasmids were carried out. By measuring OD at different inducer concentrations and having established a relationship between the concentration of inducer and gene expression, this allows for thorough characterisation of the growth slowing genes and the effect they produce on growth rate depending on how much they are expressed. See the expected behaviour in Figure 8.

Part of our future perspective includes obtaining a set of final constructs that combine an Anderson promoter with one GS gene. The previously obtained parameters on gene expression and effect on OD allowed us to calculate the relative gene expression of our genetic system and relate this parameter with growth rate. For this purpose, the proposed general structure of the set of plasmids for growth control included a combination of an Anderson promoter, an RBS and one GS gene (Figure 9). The total number of constructs obtained was 35, including all the combinations of the 5 selected Anderson promoters and the 7 GS genes.

Determination of Relative Gene Expression

Using the set of generated constructs, the expression and function of our growth control genetic system were tested as follows:

A. We measured the GFP fluorescence when the corresponding GFP was expressed under different relative strength Anderson promoters to determine a relationship between the relative strength of the Anderson Promoters, the amount of GFP they produce, and what concentration of IPTG this equals to (Figure 10). Promoter strength is shown in each case.

B. We also used the previous data gathered during the calibration stage to model what the growth rate of E. coli would be if the GSs were expressed by constitutive Anderson Promoters of different specific strengths (Figure 11). Promoter strength is shown in each case.

The overall strategy to monitor the expression and the effect on E. coli growth of our genetic system is stated within Biology and Notebook in more detail, but a 96 well plate reader was used to measure OD (600nm) and fluorescence (Ex λ 485 and Em λ 510). Anderson promoters have constitutive expression so the GS gene is also constitutively expressed, which results in a decrease in the growth rate. The final decrease in bacterial growth rate will depend on the promoter strength and the GS gene used.

Required Input

Biological modelling requires definition of the known parameters of the considered elements in our system (such as Anderson promoters' strength) and the relationship between them to generate a computational model. In addition, experimental data gathered from our laboratory work was integrated to obtain an enhanced model that better represented the real system. The integrated data is listed as follows:

Optical Density of E. coli cultures

The main input parameter constantly monitored during our experiments was the Optical Density (OD) values of the culture media using a 600nm light beam, as the standard physical property to measure bacterial growth. OD can be defined as the negative of the logarithm (base 10) of light transmission, where the transmission varies between 0 and 1 (Figure 13). OD is calibrated at the beginning of the culture with fresh culture media as blank and it increases as more cell numbers are present because of a decrease in light transmission. $$\begin{equation}\text{OD} = -\log_{10}\left(\frac{I}{I_0}\right)\end{equation}$$

After bacteria inoculation in a fresh culture, OD was followed through the different bacterial growth phases: Lag, Exponential (or Log) and Stationary (Figure 14). The monitoring of the OD over time in a bacterial culture follows a sigmoidal curve. The duration of the full cycle depends on the bacterial replication time, being about 20 minutes in E. coli. For data analysis growth rate was defined as the change of OD over time, during the exponential phase. Maximum OD was also measured in each culture corresponding to when the culture reached maximum cell density. An additional phase can be present at the end of the growth cycle (Death or Decline phase) but these values were not considered in our experiments.

Fluorescence Intensity

Fluorescence intensity measurement parameters depends on the nature of the fluorescent protein employed. For our experiments, the measurements were taken considering the values for the maximum excitation and emission of the vsfGFP protein, which are 485nm and 510nm, respectively (Figure 14).

Expression Inducer Concentration will be the main input variable in our system. IPTG (in mM) was the molecule used during the calibration stage. For other potential applications (such as drug or antibiotic effects) any other molecule can be used. The genetic system requires to be inducible by each of these molecules.

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Protocols

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Competent Cells

DH5α strain of Escherichia coli was used for DNA amplification in vivo and BL21 LEMO strain was used as the protein expression strain. Heat shock transformations were carried out using this protocol. Initially, the iGEM protocol for competent cell production was used. However, due to a low transformation efficiency, it was changed to a protocol provided by the Perak Lab team as of the 22nd of August, and all subsequent transformations were in cells that resulted from this method.

Initial competent cell protocol
Perak Competent Cell Protocol

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Agar Plates and Overnight Cultures

LB agar (2.5% Millers) for 25 mL plates, or LB for 5 mL overnights (2.5% Millers) was mixed with either ampicillin (100 ng /μL), kanamycin (50 ng /μL) and chloramphenicol (25 ng /μL) dependent upon the antibiotic resistant gene present in the plasmid transformed into E. coli.
Agar plates were grown at 37 C overnight, and liquid overnights were grown at 37 C and 220 RPM overnight.

Overnight Culture and LB Broth Protocol

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DNA Resuspension

DNA from the iGEM distribution kits was resuspended following iGEM's protocol.
Primers and gBlocks from Integrated DNA Technologies (IDT) were resuspended to 100 µM and 10 ng /µL respectively, following their advice.

iGEM Distribution Kit Resuspension Protocol
IDT Primer Resuspension Protocol
IDT gBlock Resuspension Protocol

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Minipreps

The Nippon GeneJet Miniprep kit was used, and protocol was followed. Solutions were left to elute for five minutes instead of two before the final centrifugation step, and elutions were made with 30 μL.

Mini-Prep Time Saver Protocol

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PCR

50 μL PCR solutions were made following New England Biolab's (NEB) Q5 High-Fidelity 2× Master Mix protocol. 1 ng from gBlocks (Integrated DNA Technologies) or 1 µL from previously miniprepped DNA was used as template, along with 2.5 μL of 10 μM primers, 25 μL of 2x Q5 High-Fidelity Master Mix, and made up to volume with autoclaved Milli-Q or DEPC-treated water from Invitrogen-ThermoFisher.

PCR Protocol

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Colony PCR

10 μL PCR solutions were made with 0.2 μL of 10 μM primers, 25 μL of 2× PCRBIO Taq DNA Polymerase, and made up to volume with autoclaved MQ or DEPC-treated water from Invitrogen-ThermoFisher. Samples of colonies from a ligation plate were taken and submerged onto the solution in the tube to use as template, and the same colonies were then scratched onto an agar plate. Every time protocol was carried out, at least 3 colonies were screened.

Colony PCR Protocol

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Gel Electrophoresis

Gel electrophoresis was carried out on 0.7%, 1% and 3% m/v agarose gels depending on BP length and gel extraction. All gels were made up using a 1× TAE buffer and dyed with either SYBR Safe (Thermo Fisher) or Midori Green Advance (Nippon Genetics). Gels were run at 100 V for one hour. A 1 kb Plus Ladder (Thermo Fisher) was used, and 6× Purple Gel Loading Dye (New England Biolabs) was added. Gel extractions were carried out using a Gel Extraction Kit (Nippon Genetics) following the standard protocol.

Gel Electrophoresis Protocol

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Heat Shock Transformations

Heat shock transformations were carried out using the iGEM transformation protocol.

iGEM Transformation Protocol

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Restriction Digests

The NEBcloner restriction digest tool was used to find the correct protocol for each restriction enzyme. BsaI-HFv2, BsmBI-v2, NcoI and DpnI (all New England Biolabs), were used at moments to perform restriction digests.

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Building Plasmid Constructs

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96-Well Plate Experiments

A 96 well plate reader recorded fluorescence (where relevant) and OD readings at 15 minute intervals, for a day (97 cycles). All wells contained 150 μL of culture. All cultures were diluted to a standardised OD of 0.05 and 20 μL of them were added to the relevant wells. The remaining 130 μL volume contained LB media and the relevant antibiotics and inducer in their respective required concentrations. See the linked file below.
The plate reader employed was a Hidex Sense Microplate Reader Type 425-311 (g/n 320-0250).

Plate Map produced, with concentrations and volumes for each well marked.

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Glycerol Stocks

Glycerol stocks for long term culture storage were made following Addgene's protocol.

Glycerol Stocks Protocol