Team:Glasgow/Project/Overview/RBS

Glasglow

RBS library

Summary

All 2015 iGEM teams have been invited to participate in the Second International InterLab Measurement Study in synthetic biology. Each lab will obtain fluorescence data for the same three GFP-coding devices with different promoters varying in strength. The objective is to assess the robustness of standard parts and the variability of measurements among different research groups using different lab techniques.

Motivation

For the Bioluminescence part of our project we used lux operon from Vibrio fischeri introduced to the iGEM for the first time by Cambridge team in 2010. The have used five lux genes for the assembly of the lux operon: luxA, B, C, D and E with luxA and luxB encoding bacterial luciferase and luxC, luxD and luxE encoding enzyme complex that synthesises tetradecanal, a substrate for the luciferase. This year were are adding sixth lux gene to the assembly – luxG which is known to encode a flavin reductase that provides reduced flavin mononucleotide for the bioluminescence reaction resulting in an enhanced light ouptput.

Initially we decided to optimise bioluminescence in E. coli by rearranging whole Lux operon and placing a defined relatively-weak (REF) ribosome binding site – B0032 – upstream of each of the six lux genes (Link to Cara’s Bioluminescence page). Taking this approach further, we thought of adjusting bioluminescence to E. coli by creating a B0032-derived ribosome binding site library for each lux gene. The idea behind this was to create a range of RBS combinations in a lux operon and therefore, generate E. coli strains of different bioluminescence intensity (FIGURE). We assumed that the most favourable RBS arrangements in lux operon should be observed in the E. coli colonies emitting the most light.

Design

For the construction of the RBS library, we used a master sequence based on the RBS B0032 (FIGURE). 4 nucleotides close to the actual ribosome binding site were randomised giving 32 different B0032-derived RBS variants. The predicted efficiency of each RBS library member was estimated using RBS Library Calculator (REF http://msb.embopress.org/content/10/6/731) for every lux gene (FIGURE with graphs). Technically, with 32 different RBS variants for each of the six lux genes, final RBS library for lux operon would have over a billion different RBS arrangements (Figure, show calculations??).

Strategy and approaches

Randomised PCR and Cloning, Cloning, Cloning

To start off with the RBS library construction, each lux gene was amplified by randomised PCR using primers with a B0032-derived master sequence for RBS. PCR products were then ligated into plasmid PSB1C3 and transformed to E. coli strain TOP10 which is a recA- mutant meaning that any unwanted gene rearrangements between chromosomal DNA and plasmid DNA can be avoided.

In order to induce expression of luxABG and luxCDE in the prospect experiments, luxA and luxC PCR products were inserted downstream the pBAD (BBA_ I0500) and R0011N (BBa_K1725080) promoters, respectively. pBAD is widely used promoter inducible by L-arabinose, in our assays we have used 1% arabinose to activate the promoter. R0011N promoter is similar to IPTG-regulated, LacI-repressed R0011 promoter (BBa_R0011) but contains an extra NheI site to make Biobrick insertion identification easier in the restriction digests. Since we have used lacI- Top10 cells for our transformations, R0011N was constitutively active.

Colonies from the transformation plates were then washed and plasmid DNA was purified and sequenced to ensure that all 32 RBS library members were present in the sample (FIGURE of sequencing). Similar approach was applied to the subsequent ligations in the assemblies of pBAD.luxABG and R0011N.luxCDE.

It is worth mentioning that before every ligation, inserts and vectors were extracted and purified from a gel stained with non-toxic blue dye Azure, which is very convenient and fast gel staining tool proposed by our team (Link to James’ Azure page) & picture of the blue gel with lux genes to be extracted.

Testing pBAD.luxAB

Once we have assembled luxA and luxB together with a pBAD promoter upstream, we wanted to determine if the construct allows cells to respond to the decanal in the environment. In addition to that, we also aimed to visualise RBS library in the construct as RBS of different strengths should cause variability in bioluminescence intensity between colonies. To start, we grew transformed cells on the L-agar with 1% arabinose and then exposed them to the 5% decanal solution. Several 10μl drops of solution were applied on the lid of the Petri dish and the lids were then immediately placed on the plates with cells and kept for a few minutes. Lids were then taken off and plates were photographed in the dark room with the 30s exposure at the ISO 64000.

As seen in the FigureX, colonies that grew on the plate containing arabinose (ara+) show bioluminescence activity in the dark while control plate with no arabinose (ara-) does not contain any bioluminescent colonies. More importantly, in the ara+ plate we observe that, for a human eye, colonies vary in bioluminescence intensity from very bright to absolutely blank colonies. Therefore, here we show that E. coli is able to uptake decanal from the environment and produces light when the expression of luxAB is turned on. Moreover, we demonstrate that some of the RBS arrangements in the pBAD.IuxAB construct are more efficient than others in terms of stimulating translation initiation. asdasdasdsakdhsuhdusfhailsdufhudshfiuasdfsdf

For further testing, we have picked up 12 colonies that exhibited different bioluminescence intensity: a range from very bright to dim or blank colonies. We tested if the construct sizes are similar in all 12 colonies by gel electrophoresis of single restriction digests (GEL Picture). As the gel results suggest, plasmid sizes in all 12 colonies are similar which allows us assume that the main difference is in the ribosome binding sites. In addition, we have made short streaks of each colony on ara+ plate in order to compare their brightness on a bigger resolution (Picture of streaks + colonies plate with tagged locations of colonies picked). From the picture we can clearly see colony F being the brightest colony on the plate and some of the colonies producing very little of visible bioluminescence. This again supports our hypothesis about some RBS combinations being more favourable by the E. coli translation machinery.

Typhoon FLA 9500 calibration

A dilution series was measured for phiLOV protein (Figure 2), converted to numerical readings (Table 1) and a calibration curve (Figure 3) carried out to calibrate the Typhoon. Fluorescent proteins derived from voltage (LOV) domains are smaller and more efficient under anaerobic conditions than green fluorescent proteins (GFP) (Buckley et, al. 2015). iLOV, an improved LOV flavoprotein, was originally engineered as a reporter for viral infection from phototropin, the blue light receptor. We used phiLOV which is a photostable version of the iLOV fluorescence reporter.

Figure 2: Fluorescence readings of a dilution series of phiLOV. 67.5µg = 67.5µg phiLOV in 100µl PBS. Each concentration was carried out twice.

Table 1: Summary of the fluorescence readings of phiLOV protein.

Figure 3: Calibration curve of fluorescence of phiLOV

Methodology

Protocol for cloning devices

The devices, as shown in Table 2, were prepared using BioBrick assembly. Parts J23101, J23106, J23117, I13504, I20270 and R0040 were taken from the iGEM distribution plates and each transformed into TOP-10 competent cells. The promoters were digested with Pst1 and Spe1 and the GFP part, I13504, was digested with Xba1 and Pst1. The I13504 part was then ligated into each promoter plasmid and transformed into TOP-10 cells to create the three required devices in pSB1C3 (Figure 4). Restreaks were carried out for one colony of each device and control and three colonies of each (labelled 1, 2 and 3) were picked and grown separately. Sequencing was carried out to check the correct devices had been created.

Table 2: Summary of BioBrick used

Figure 4: Device cloning strategy

Preparation for measurements

Overnight cultures of colony 1-3 of each device were set up (in Luria broth with chloramphenicol) to provide 1ml for measuring on a 96-well plate. As the he broth gave noticeable background fluorescence samples were also prepared by spinning down cells, in the overnight cultures, to pellets and resuspending in PBS (phosphate buffered saline). It was determined the PBS method gave the most accurate measurements so readings were taken using this method for all three biological replicates and technical replicates.

The recipe used for a 1 x solution of PBS was 8g NaCl, 0.2g KCl, 1.44g Na2HPO4 and 0.24g KH2PO4 dissolved in 800ml of H2O, the pH adjusted to 7.4 and the final volume made up to 1 litre with distilled H2O.

Protocol for measurements

The spectrometer was used to measure absorbance at 600nm of each sample. Samples were then diluted to 0.5 with PBS and rescanned. The Typhoon was used to measure the GFP fluorescence at 475nm of each device and control on a 96 well plate. These methods were repeated for each biological and technical replicate.

The controls

A negative control for background cell fluorescence was included as cells containing the device R0040 but without a promoter, to mimic burden of the promoter. A positive control for GFP fluorescence was included as cells containing the device I20270, a GFP part with the promoter J23151. PBS was used to control for media-only background. In addition in order to obtain absolute values for fluorescence, set standards of FAM oligo were also measured.

Protocol for calculating a conversion factor for absolute units

We used a 6-FAM (6-carboxyfluorescein) labelled oligonucleotide to standardise our fluorescent results. This allowed us to express our GFP levels as equivalent amounts of 6-FAM. 6-carboxyfluorescein is the most commonly used fluorescent dye for labelling oligonucleotides, and therefore should be readily available to most iGEM teams. 6-FAM labelled oligonucleotides can be quantitated by measuring the UV absorbance at 260 nm (measuring the DNA concentration). 6-FAM has similar fluorescent properties to eGFP (excitation peak at 492 nm and an emission maximum of 517 nm for 6-FAM compared to 488 nm excitation and 508 nm emission for E0040 GFP mut3b).

A dilution series of FAM labelled oligonucleotide was measured (Figure 5) and converted to numerical readings (Table 3) to enable absolute values for the devices to be calculated. The calibration curve (Figure 6) has a line gradient of 4.79x10^6. Therefore the fluorescence readings of the devices will be divided by the conversion factor of 4,790,000 to give absolute fluorescence as equivalent to pmol of FAM labelled oligonucleotide. Absolute values should be comparable across different equipment and protocols.

Figure 5: Fluorescence readings of a dilution series of FAM labelled oligonucleotide. 10pmol = 10pmol FAM labelled oligonucleotide in 100µl PBS.

Table 3: Fluorescence readings of FAM labelled oligonucleotide.

Figure 6: Confirmation of linear relationship between FAM labelled oligonucleotide concentration and measured fluorescence on the Typhoon. Gradient of this calibration curve is the conversion factor for fluorescence as measured by the Typhoon to equivalent pmol of FAM labelled oligo.

Measurements

Direct Measurement (Raw Data)

The A600 of each device colony 1-3 and technical replicates were measured along with the controls (table 4).

Table 4: Absorbance at 600nm for each biological and technical replicates of the devices and controls. Units are arbitrary.

The fluorescence was also measured (figure 7) and the resulting images converted to numerical readings (table 5).

Figure 7: Fluorescence results of the three devices and the positive and negative controls. A. Shows the image at low brightness to compare the J23101 and J23106 devises. B. Shows the image at high brightness to compare the J23117 device with the two brighter devices.

Table 5: Summary of fluorescence data measured for the three devices and controls.

Derived Measurements (Conversion to Absolute units)

1. The average background absorbance was removed by subtracting the average of the empty wells with no PBS or sample (423,343.279).
2. The average absorbance of control E.coli cells was removed by subtracting the average of the TOP 10 cells with R0040 (222,475).
3. These values were divided by the absorbance values at 600nm to give the fluorescence per OD 600 in arbitrary units (Table 6).
4. Dividing these values by the conversion factor as determined from the FAM oligo dilutions (479,000) gives the absolute fluorescence equivalent to pmol of FAM oligo per A600 of cells (Table 6).

Table 6: Derived measurements of devices and controls.

Figure 8:

Estimation of absolute number of GFP molecules per cell

We attempted to estimate the absolute number of GFP molecules per cell (Table 7) using our phiLOV results and some simplifying assumptions.

Table 7: Summary of absolute number of GFP molecules per cell.

In order to estimate the absolute number of GFP molecules per cell the following calculations were carried out:

ilov stock = 1.35 mg/ml = 1.35 g/l

MW = approx. 150x110 = 16500

ilov stock = 82uM

Avogadro’s number = 6.02x10^23
⇒ Diluted stock 2 fold and used 100 ul in well = 1/20000 litre = 4.1 nmoles
⇒ 4.1 nmoles of phiLOV gave a reading of 490,000,000

J23101:I13504

⇒ 200ul cells at A600 of 1.0 with J23101 promoter gave fluorescence reading of average 1,000,000,000.

So 200 ul cells equivalent to 4.1 *1000/490 = 8.4 nmoles iLOV

⇒ GFP approx. 11.5 x brighter than iLOV per mol

So 8.4 nmoles iLOV gives equivalent fluorescence to 8.4/11.5 = 0.73 nmoles of GFP per 200 ul cells
So 200 ul cells contains 0.73x10^-9 x (Avogadro’ s number 6.02x10^23) = 4.4x 10^14 molecules

⇒ Fluorescence was quoted for cells at OD600 = 1.0
⇒ 1 OD600 of TOP10 = 4x10^8 cells per 200 ul

So 1 cell contains 4.4 x 10^14 / 4 x10^8 = 1 million copies of GFP

⇒ 1 million molecules of GFP = (1000000/6.02x10^23) = 1.7x10^-18 moles GFP

27,000 x 1.7 x 10^-18 = 4.7 x 10^-14 g = 47 femto grams

⇒ typical total protein content of bacterial cell = 100 femto grams

So approximately half of all cellular protein is GFP

J23106:I13504

⇒ 200ul cells at A600 of 1.0 with J23106 promoter gave fluorescence reading of average 250,000,000.

So 200 ul cells equivalent to 4.1 *25/49 = 2.09 nmoles iLOV

⇒ GFP approx. 11.5 x brighter than iLOV per mol

so 0.209 nmoles iLOV gives equivalent fluorescence to 2.09/11.5 = 0.181 nmoles of GFP per 200 ul cells so 200 ul cells contains 0.181x10^-9 x (Avogadro’ s number 6.02x10^23) = 1.08x 10^14 molecules

⇒ Fluorescence was quoted for cells at OD600 = 1.0
⇒ 1 OD600 of TOP10 = 4x10^8 cells per 200 ul

So 1 cell contains 1.08x10^14 / 4 x10^8 = 272,405 = 270,000 copies of GFP

⇒ 270,000 molecules of GFP = (270,000/6.02x10^23) = 4.48x10^-19 moles GFP

27,000 x 4.48x10^-19 = 1.2 x 10^-14 g = 12 femto grams

⇒ typical total protein content of bacterial cell = 100 femto grams

so 12% of cellular protein is GFP

J23117:I13504

⇒ 200ul cells at A600 of 1.0 with J23117 promoter gave fluorescence reading of average 2,500,000

So 200 ul cells equivalent to 4.1 *25/4900 = 0.0206 nmoles iLOV

⇒ GFP approx. 11.5 x brighter than iLOV per mol

so 0.0206 nmoles iLOV gives equivalent fluorescence to 0.0206/11.5 = 1.79x10^-3 nmoles of GFP per 200 ul cells
so 200 ul cells contains 1.79x10^-12 x (Avogadro’ s number 6.02x10^23) = 1.08x 10^12 molecules

⇒ Fluorescence was quoted for cells at OD600 = 1.0
⇒ 1 OD600 of TOP10 = 4x10^8 cells per 200 ul

So 1 cell contains 1.08 x 10^12 / 4 x10^8 = 2,700 copies of GFP

⇒ 1 million molecules of GFP = (2,700/6.02x10^23) = 4.48x10^-21 moles GFP

27,000 x 4.48x10^-21 = 1.2 x 10^-16 g = 0.12 femto grams

⇒ typical total protein content of bacterial cell = 100 femto grams

so 1.2x10^-3 % of all cellular protein is GFP

References

Buckley, A. Petersen, J. Roe, A. Douce, G. Christie, J. (2015). LOV-based reporters for fluorescence imaging. Current Opinion in Chemical Biology. 27 (1), p39–45.