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.

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.

Inviting Mr. Bright to the party: luxG

As mentioned before, LuxG is a known flavin reductase and provides reduced flavin mononucleotide to luciferase LuxAB resulting in increased bioluminescent activity. Therefore, having the pBAD.luxAB construct ready we have inserted luxG gene downstream the luxAB in order to increase the bioluminescence in cells. Transformed cells were grown on both ara+ and ara- plates and treated with decanal as described earlier.

As can be seen in the FigureX, only colonies that have grown on the ara+ plates exhibit luminescence while control plates remain blank in the dark. Similarly to the results observed in pBAD.luxAB testing, here we also see a range of luminescence between colonies on ara+ plate most likely corresponding to the different ribosome binding sites. However, on the ara+ plates we observe only half as many colonies as on the ara- plates suggesting luxG possibly having a negative role for cell growth. To test this, we have picked 136 random colonies from the ara- plate and streaked them on new ara+ and ara-plates to see difference in survivability. Results are presented in the FigureX where short streaks appear to be similar on both ara+ and ara- plates with no bacterial growth disruption on the ara+ plate. Therefore, we assume that the negative luxG effect is not very severe and may be only observable on the transformation plates where colonies are formed from a single cell.

Additionally, in order to compare bioluminescence between cells where luxG is expressed and in cells where it is not, we also streaked previously described 12 pBAD.luxAB colonies and 12 brightest pBAD.luxABG colonies on the same ara+ plate. As can be seen from the FigureX, most of the pBAD.luxABG colonies appear to exhibit brighter luminescence than the pBAD.luxAB colonies which is what we expect to see.

We have picked up the brightest pBAD.luxABG colony, determined RBS for each gene by sequencing and submitted the construct as the biobrick BBa_K17252340.

Story about luxCDE

TSimultaneously with the assembly of pBAD.luxABG we have also assembled R0011N.luxCDE construct using similar approaches. In order to test our RBS library in luxCDE we employed two strategies. Firstly, we wanted to test if two cells, one expressing luxABG and other expressing luxCDE, are able to produce luminescence when mixed and if so, does the bioluminescence depend on the amount of produced tetradecanal. We picked up 24 random R0011N.luxCDE colonies from the transformation plate and grew them overnight in the eppendorf shaker. The following day we mixed equal volumes of 24 R0011N.luxCDE and pBAD.luxABG (BBa_K17252340) overnight cultures in a 96-well plate. pBAD.luxABG overnight culture was added with 1% arabinose and grown for two hours in order to induce pBAD promoter. 96-well plate was then photographed and no visible bioluminescence was observed.

Paragraph on generating strain with luxABG in PSB1C3 and luxCDE in PSB3K3

Results

- Cell-cell comunication
- Decanal experiments
- Spectrum experiments and comparison to Cambridge operon