From 2010.igem.org

Characterized BioBricks

We entered the following BioBricks to the partsregistry:

<groupparts>iGEM010 Bielefeld-Germany</groupparts>

<partinfo>K238008</partinfo>: virA

We wanted to use this part in our project, but could only obtain unexpected/faulty restriction patterns. Finally we chose to sequence the part, hoping to find the cause for the maintained restriction patterns. Unfortunately we could not approve the sequence of <partinfo>BBa_K238008</partinfo> deposited in the parts registry so that we chose to design our own VirA BioBrick. I strongly recommend using our VirA since it has been approved by multiple means, e.g. restriction patterns and sequencing (<partinfo>K389001</partinfo>).

<partinfo>BBa_K238011</partinfo>: vir-promoter

We made a restriction analysis and sequenced parts of this BioBrick.

<partinfo>P1010</partinfo>: ccdB-gene

The ccdB gene targets the gyrase of Escherichia coli and is lethal for all E. coli strains without the gyrase mutation gyrA462 [1]. The ccdB BioBrick is used for the 3A-assembly as a positive selection marker. We transformed this BioBrick into E. coli JM109, DH5α, TOP10, XL1-Blue, EC100D and DB3.1. E. coli JM109, XL1-Blue and DH5α seem to be ccdB resistant because there were as much colonies after P1010 transformation as observed with DB3.1. The P1010 works as expected in E. coli TOP10, EC100D (no colonies after transformation) and DB3.1 (many colonies after transformation).

It seems that not only the gyrase mutation gyrA462 is causing a ccdB resistance. Also the gyrase mutation gyrA96 gives E. coli a ccdB resistance. This should be kept in mind when assembling BioBricks with the 3A assembly.

<partinfo>K389004</partinfo>: Luciferase from pGL4.10[luc2]

<partinfo>K389011</partinfo>: VirA screening device

Ratio of surviving colonies of E. coli EC100D carrying unmutated <partinfo>K389010</partinfo> and <partinfo>K389011</partinfo> plated on PA agar plates with chloramphenicol, ampicillin and different concentrations of kanamycin. Comparison between cells that were induced with acetosyringone with cells that were not induced.

The ratio of surviving colonies ϕS was calculated like

with the number of colony forming units CFU, the concentration of kanamycin on the considered plate KanX and no kanamycin on the plate Kan0.

<partinfo>K389012</partinfo>: VirA reporter system with luciferase

coming more soon

<partinfo>K389015</partinfo>: VirA/G reporter device with luciferase

Growth functions and Luciferase expression for <partinfo>K389015</partinfo>

To characterize this part we performed several cultivations with different concentrations of [http://www.chemblink.com/products/2478-38-8.htm acetosyringone] as inducer and measured the luminescence emitted by the luciferasereaction with Luciferin (Protocol). We used Escherichia coli DB3.1 carrying the pSB1C3::K389015 plasmid. Even without inducer the bacteria carrying the plasmid showed decelerated growth. In addition acetosyringone affected the growth rates (we used a stocksolution of 20 mM acetosyringone solved in 10 % (v/v) DMSO). Growth curves, averaged specific growth rates and doubling times are shown below. It can be observed, that E. coli carrying the pSB1C3::K389015 plasmid growths nearly linear.

Fig. 1: Growth Curves for E. coli DB3.1 without plasmid and carrying <partinfo>K389015</partinfo> with different acetosyringone concentrations in LB medium with 10 mg ml^-1 chloramphenicol.

The specific growth rates µ and doubling times t_d are calculated with the OD₆₀₀ and following formulas:

Exemplary induction curves with the luminescence normalized to OD₆₀₀ are shown in Fig. 2. We observed a basal transcription, but the induction with acetosyringone is undoubtedly. The detailed data analysis and transfer function is described below.

Fig. 2: Induction curves for E. coli DB3.1 carrying <partinfo>K389015</partinfo> with different acetosyringone concentrations in LB medium with 10 mg ml^-1 chloramphenicol. The relative luminescence units (RLU) are normalized to OD₆₀₀ and plotted against the cultivation time in h

Transfer function

The data for the transfer function was measured and analyzed as described below. The data was fitted with a dose response function of the form

with the Hill coefficient p, the bottom asymptote A1, the top asymptote A2 and the switch point log(x₀). Figure 3 shows the measured ratio ɸ_I between induced (i) and uninduced (u) relative luminescence units (RLU) per OD₆₀₀ plotted against the logarithm of the concentration of the inductor [http://www.chemblink.com/products/2478-38-8.htm acetosyringone] in µM. The fit has an R² = 0.98.

Fig. 3: Transfer function for the part <partinfo>K389015</partinfo> (R² = 0.98).

The important data from the transfer function is summarized in table 1:

The fully induced VirA/G signaling system with luciferase read-out has a 2.2 fold increased expression compared to the uninduced system. The Hill coefficient is > 1, so a positive cooperation can be observed ([http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6WMD-4V42JG5-1&_user=10&_rdoc=1&_fmt=&_orig=search&_sort=d&_docanchor=&view=c&_acct=C000050221&_version=1&_urlVersion=0&_userid=10&md5=b6431553217aca1129c5b441f4b78425 D Chu et al., 2009]). The [http://partsregistry.org/Switch_Point switch point] of the system is at about 32 µM, so this is the concentration at which the device output is 50% of the maximum output.

Response time

The system needs at least one hour to show a measurable reaction to an induction with acetosyringone. In the following illustration the reaction of the system to induction with 200 µM acetosyringone in the exponential growth phase is shown. For a good separation of the induced system from the uninduced system at least two hours are needed.

Fig. 4: Cultivation of <partinfo>K389015</partinfo> in Escherichia coli DB3.1. Induction with 200 µM acetosyringone during the exponential growth phase.

Data Analysis

Because the luciferase accumulation is very different in different cultivations, the uninduced negative control was used as internal standard. To show the behaviour of the VirA/G signaling system when induced, the ratio ɸ_I between induced (i) and uninduced (u) relative luminescence units (RLU) per OD₆₀₀ is calculated:

As seen above, at least one hour is needed to separate the induced luminescence signal from the uninduced, so ɸ_I > 1. Within a cultivation ɸ_I is rising during the first hours and is decreasing after it reached a maximum at OD₆₀₀ ~ 1. This is shown in figure 3:

Fig. 5A: Typical development of ɸ_I plotted to OD₆₀₀ of a cultivation with <partinfo>K389015</partinfo> in E. coli DB3.1 with polynomial fit (5th order, R² = 0.75, induced with 200 µM acetosyringone). Fig. 5B: Another typical development of ɸ_I plotted to OD₆₀₀ of a cultivation with <partinfo>K389015</partinfo> in E. coli DB3.1 with polynomial fit (5th order, R² = 0.87, induced with 400 µM acetosyringone).

To measure the ratio of increasing promoter activity by inducing the system ɸ_I samples for analyzation should be taken at OD₆₀₀ = 1 +/- 0.5. The highest ɸ_I in this range of the cultivation is taken for the calculation of the transfer function.

Plasmid conformation analysis

A plasmid conformation analysis for the BioBrick <partinfo>K389015</partinfo> in <partinfo>pSB1C3</partinfo> was performed by the [http://web.plasmidfactory.com/de/ PlasmidFactory] by Capillary Gel Electrophoresis (CGE). The chromatogram is shown in fig. 6 and the results in tab. 3. The data shows a high percentage of covalently closed circular (ccc) plasmid DNA. This is the biological active shape of plasmids so a high percentage of ccc plasmid DNA indicates a high quality of plasmid DNA ([http://web.plasmidfactory.com/en/service_CGE.html PlasmidFactory]).

Fig. 6: Chromatogram of the CGE of the BioBrick <partinfo>K389015</partinfo> in <partinfo>pSB1C3</partinfo> performed by the [http://web.plasmidfactory.com/de/ PlasmidFactory] (Bielefeld).

<partinfo>K389016</partinfo>: VirA/G reporter device with mRFP

Protocols for Cultivation and Measurement

Growth functions and mRFP expression for <partinfo>K389016</partinfo>

To characterize this part we performed several cultivations with different concentrations of acetosyringone as inducer and measured the fluorescence emitted by mRFP (Protocol). We used Escherichia Coli DB3.1 carrying the pSB1C3::K389016 plasmid. Even without inducer the bacteria, carrying the plasmid showed decelerated growth. In addition acetosyringone affected the growthrates (we used a stocksolution of 20 mM acetosyringone solved in 10 % (v/v) DMSO). Growth curves, averaged specific growth rates and doubling times are shown below. It can be observed, that E.coli carrying the pSB1C3::K389016 plasmid growths nearly linear.

Fig. 1: Growth Curves for E. coli DB3.1 without plasmid and carrying <partinfo>K389016</partinfo> with different acetosyringone concentrations in LB medium with 10 mg ml^-1 chloramphenicol.

The specific growth rates µ and doubling times td are calculated with the OD600 and following formulas:

Exemplary induction curves with the fluorescence normalized to OD600 are shown in Fig.2. We observed a basal Transcription, but the Induction with acetosyringone is undoubtedly. The detailed data analysis and transfer function is described below.

Fig. 2: Induction Curves for E. coli DB3.1 carrying <partinfo>K389016</partinfo> with different acetosyringone concentrations in LB medium with 10 mg ml^-1 chloramphenicol. The relative fluorescence units are normalized to OD600 and plotted against the ime in h

Transfer function of <partinfo>K389016</partinfo>

The data for the transfer function was measured and analyzed as described below. The data was fitted with a dose response function of the form

with the Hill coefficient p, the bottom asymptote A1, the top asymptote A2 and the switch point log(x₀). Figure 1 shows the measured normalized specific production rates q_P,n (eq. 8) plotted against the logarithm of the concentration of the inductor [http://www.chemblink.com/products/2478-38-8.htm acetosyringone] in µM. The fit has an R² = 0.99.

Fig. 1: Transfer function for the part <partinfo>K389016</partinfo> (R² = 0.99).

The important data from the transfer function is summarized in table 1:

So the fully induced VirA/G signaling system has a 2.6 fold increased expression compared to the uninduced system. The Hill coefficient is > 1, so a positive cooperation can be observed ([http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6WMD-4V42JG5-1&_user=10&_rdoc=1&_fmt=&_orig=search&_sort=d&_docanchor=&view=c&_acct=C000050221&_version=1&_urlVersion=0&_userid=10&md5=b6431553217aca1129c5b441f4b78425 D Chu et al., 2009]). The [http://partsregistry.org/Switch_Point switch point] of the system is at about 25 µM, so this is the concentration at which the device output is 50% of the maximum output.

Data analysis for <partinfo>K389016</partinfo>

The data analysis is made in three steps. First step is the processing of the fluorescence raw data gained by the fluorescence plate reader for every sample:

In the second step the RFU_corrected of every sample is plotted against the cultivation time it was drawn. The data is fitted by an exponential fit of the following style:

The accumulation of mRFP in the cells is always exponential. A typical fitted product accumulation curve is shown below:

Fig. 2: Exponential fit on the measured RFU plotted against cultivation time of a cultivation of <partinfo>K389016</partinfo> in Escherichia coli DB3.1 in LB medium with 10 mg ml^-1 chloramphenicol and 150 µM acetosyringone.

The product accumulation in a cultivation can be described as:

with the amount of product P, the cell count X and the specific production rate q_P.

RFU is commensurate to the concentration of mRFP (P) and the OD₆₀₀ is commensurate to the cell count (X) ( Canton and Labno, 2004):

With these assumptions it is possible to calculate the specific production rate of mRFP q_P in the third step: the specific production rate for every sample of a cultivation is calculated by the derivation of the exponential fit line which describes the accumulation of product in the culture (dRFU/dt) and the measured OD₆₀₀ data:

The specific production rates q_P of all samples of all cultivations made with a specific inductor concentration c are averaged and normalized against the specific production rate of the uninduced system q_P,0:

This normalized specific production rate we calculated is commensurate to relative promotor units (RPU) which is commensurate to PoPS (polymerase per seconds) ([http://partsregistry.org/Part:BBa_F2620:Experience/Endy/Data_analysis Canton and Labno, 2004]; [http://partsregistry.org/Part:BBa_J23101:Experience Pasotti et al., 2009]):

Control of BioBrick quality by capillar gel electrophoresis (GCE)

The representative tested BioBrick shows ccc-type of over 91 %, meaning pDNA quality is optimal.

Standard CGE Analysis Sample ID: K389016 Lauf: 10/22/2010 12:55:04 PM Report: 10/25/2010 10:03:46 AM PlasmidFactory GmbH & Co KG

<partinfo>K389052</partinfo>: tightly regulated lac operon with mRFP readout

Fails...more soon.

<partinfo>K389421</partinfo>, <partinfo>K389422</partinfo>, <partinfo>K389423</partinfo>: Sensitivity Tuner amlified Vir-test system

By self designed PCR-Primer we excluded terminal GFP and the initial promoter pBAD/araC, for replacing our own VirB promotor and reporter gene luc (luciferase). Primers were designed for sensitivity tuner [http://partsregistry.org/Part:BBa_I746370 I746370], [http://partsregistry.org/Part:BBa_I746380 I746380] and [http://partsregistry.org/Part:BBa_I746390 I746390] so that standard assembly would be possible. Assembling of PCR-products took place by Silver Assembly.

Accomplishment

PCR-Primer Design

Primer forward activator phage P2:

5`-GTT TCT TCG AAT TCG CGG CCG CTT CTA GAT GTT TCA TTG TCC TTT ATG CC-3`

Primer forward activator phage PSP3:

5`-GTT TCT TCG AAT TCG CGG CCG CTT CTA GAT GAT GCA CTG CCC GTT ATG- 3`

Primer forward activator phage phi R73:

5`-GTT TCT TCG AAT TCG CGG CCG CTT CTA GAT GCG CTG CCC TTT CTG-3`

Primer backward Promotor PF from phage P2:

5`-GTT TCT TCC TGC AGC GGC CGC TAC TAG TAT TTC TCC TCT TTC TCT AGT AAG TGG- 3`

Characterization tests

Cultivation was done by induction with Acetosyringone at 50 µM. Controls were not induced Sensitivity Tuner devices as well as induced and not induced nativ system ([http://partsregistry.org/Part:BBa_K389015 K389015]; without tuning elements). Induction was done upon inoculation. Measuring point for amplification factor calculation was OD 1.0.

Results

Three sensitivity tuned Vir-Gen sensing systems were obtained: [http://partsregistry.org/Part:BBa_K389421 K389421], [http://partsregistry.org/Part:BBa_K389422 K389422] and [http://partsregistry.org/Part:BBa_K389423 K389423] distinguishing by the amplification level of luc transcription.

Figure 1: Amplification factor of induced, 50 µM Acetosyringone (red) and not induced (green) modified Sensitivity Tuner K389421, K389422 and K389423, Standard deviation shown.

The amplification factor was received by apply [http://partsregistry.org/Part:BBa_K389015 K389015] as reference. Amplification calculation was done by normalizing relative luminescence units emitted from luciferase per OD. Output-signal amplification is in the induced contructs (red) [http://partsregistry.org/Part:BBa_K389422 K389422] and [http://partsregistry.org/Part:BBa_K389423 K389423] 100 and respectively 200 fold higher than in not induced controls (green). An exception is K389422 were induced and not indiced system revealed analog results. Corresponding to data of iGEM Team, Cambridge 2009, K389423 (originated from [http://partsregistry.org/Part:BBa_I746390 I746390]) shows the highest amplification rate of all tested Sensitivity Tuners. Our results indicate to higher amplification rate of [http://partsregistry.org/Part:BBa_K389421 K389421] than [http://partsregistry.org/Part:BBa_K389422 K389422] of 100 fold under induced conditions. The controls also show high basal transcription rates.

Because there is small difference in induced and not induced system visible and basal transcription rates are high, we assume that the sensitivity tuning constructs are not well applicable for luciferase measurements.

For further theory click Read out system

Sequenced BioBricks

Own BioBricks

The sequencing of the following of our own BioBricks was succesful and lead to the expected results:

• <partinfo>K389001</partinfo> (not fully completed)

• <partinfo>K389002</partinfo>

• <partinfo>K389003</partinfo>

• <partinfo>K389004</partinfo>

• <partinfo>K389005</partinfo>

• <partinfo>K389010</partinfo> (not fully completed)

• <partinfo>K389011</partinfo> (not fully completed)

• <partinfo>K389012</partinfo> (not fully completed)

• <partinfo>K389013</partinfo> (not fully completed)

• <partinfo>K389015</partinfo> (not fully completed)

• <partinfo>K389016</partinfo> (not fully completed)

• <partinfo>K389050</partinfo>

Other BioBricks

• <partinfo>K238008</partinfo> sequencing gave negative results - infos in registry are not correct!

• <partinfo>K238011</partinfo> sequencing gave negative results - infos in registry are not correct!

References

[1] http://openwetware.org/wiki/CcdB, CcdB (seen on 10.10.10).

[2] http://openwetware.org/wiki/E._coli_genotypes, E. coli genotypes (seen on 10.10.10).

[3] Metcalf, W.W. et al. (1994) Gene 138, 1.

[4] Stadler J, Lemmens R, Nyhammar T 2004, Plasmid DNA purification, The J. of Gene Medicine,Vol.6, pp.54–S66

[5] Behrens B, Eppendorf AG, Laborpraxis, Nr.20, Reinste Plasmid-DNA in nur 9 Minuten.

Canton B and Labno A (2004) [http://partsregistry.org/Part:BBa_F2620:Experience/Endy/Data_analysis Data processing of Part BBa_F2620].

Chu D, Zabet NR, Mitavskiy B (2009) [http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6WMD-4V42JG5-1&_user=10&_rdoc=1&_fmt=&_orig=search&_sort=d&_docanchor=&view=c&_acct=C000050221&_version=1&_urlVersion=0&_userid=10&md5=b6431553217aca1129c5b441f4b78425 Models of transcription factor binding: Sensitivity of activation functions to model assumptions], J Theor Biol 257(3):419-429.

Pasotti L, Zucca S, Del Fabbro E (2009) Characterization experiment on BBa_J23100, BBa_J23101, BBa_J23118, http://partsregistry.org/Part:BBa_J23101:Experience.