Team:Glasgow/Project/Overview/RBS
Glasglow
RBS library
Summary
Aim
To optimise bioluminescence in Escherichia coli by creating a range of Ribosome Binding Sites (RBS) for each of the six genes in the luxCDABEG operon from Aliivibrio fisheri, originally submitted to the registry as a single BioBrick (K325909) in 2010 by the Cambridge team.
Results
- Designed RBS library with 32 variants for each lux gene - Made luxABG and luxCDE constructs from the RBS library – over 1000 RBS variantions for each construct - Showed that cells are able to uptake decanal from the environment and produce light when luxAB or luxABG is expressed - Visualised RBS library for luxAB, luxABG and luxCDE constructs and determined optimal RBS arrangements for E. coli
Biobricks
Documented and submitted:
- BBa_K1725340: I0500-RBS-luxA-RBS-luxB-RBS-luxG
- BBa_K1725341: I0500-RBS-luxA-RBS-luxB-RBS-luxG-K1725080-RBS-luxC-RBS-luxD-RBS-luxE
- BBa_K1725301-BBa_K1725332: RBS library
- BBa_K1725342: K1725080-RBS-luxC-RBS-luxD-RBS-luxE (High decanal production)
- BBa_K1725343: K1725080-RBS-luxC-RBS-luxD-RBS-luxE (Low decanal production)
Motivation
For the Bioluminescence part of our project we used the luxCDABEG operon from A. fischeri introduced to the iGEM by Cambridge team in 2010. Five lux genes are known to be essential for the bioluminescence production: luxA and luxB encoding bacterial luciferase and luxC, luxD and luxE encoding enzyme complex that synthesises tetradecanal, a substrate for the luciferase. Sixth gene, luxG encodes 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, as described on our Bioluminescence page. Taking this approach further, we thought of adjusting bioluminescence in 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. 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 1). 4 nucleotides within 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. Theoretically, 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.
Strategy and approaches
Randomised PCR and Cloning, Cloning, Cloning
For construction of the RBS library, 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, luxA and luxC PCR products were inserted downstream the pBAD (BBA_ I0500) and R0011N (BBa_K1725080) promoters, respectively. pBAD is a 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 2). A similar approach was applied to the subsequent ligations in the assemblies of pBAD.luxABG and R0011N.luxCDE.
Figure 2
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. The method of decanal application that we developed involved placing 6 decanal drops of 10μl on the lid of the Petri dish with a pipette, immediately putting lids on the plates with cells and then letting the decanal to diffuse for 3-4 minutes. Plates without the lids were then photographed in the dark room at the 30s exposure and ISO 64000. We have also found out that leaving lids with decanal on the plates result in much intense bioluminescence visible by naked eye.
Figure 3
As seen in the Figure 3, 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 produce light when the expression of luxAB is turned on. Moreover, we demonstrate that some of the RBS arrangements in the pBAD.luxAB construct are more efficient than others in terms of stimulating translation initiation.
For or further testing, we have picked up several colonies that exhibited different bioluminescence intensity: a range from very bright to dim or blank colonies. We tested all chosen colonies by restriction digests for the plasmid size in order to test if any of the genes are missing from the dim colonies (Figure 4). From the gel results all colonies appear to be similar in plasmid size allowing us to assume that the main difference between them 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 (Figure 5). 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.
Figure 5
Mr. Bright: 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.
Figure 6
As can be seen in the Figure 6, 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.
Figure 7
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 Figure 8, most of the pBAD.luxABG colonies appear to exhibit brighter luminescence than the pBAD.luxAB colonies which is what we expect to see.
Figure 8
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
Simultaneously 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 have mixed equal volumes of 24 R0011N.luxCDE and pBAD.luxABG (BBa_K17252340) overnight cultures in a 96-well plate (pBAD.luxABG overnight culture was grown for two hours in 1% L-arabinose before addition to the plate). However, we could not observe any bioluminescence in the wells.
We have repeated the assay, this time growing both R0011N.luxCDE and pBAD.luxABG overnight cultures together in 1% L-arabinose for several time intervals. We only tested two random R0011N.luxCDE colonies and after 3hrs we observed a very low level of bioluminescence which was different between two samples (Figure 9). This was a first proof that decanal is released by cells expressing luxCDE and can then be uptaken by cells with luxABG genes. However, due to the lack of time we did not study this process in more detail leaving it for the possible future investigations.
Figure 9
The second strategy that we have developed for testing our RBS library in luxCDE assembly involved generating E. coli strain with two plasmids: PSB1C3 with pBAD.luxABG and PSB343 with R0011N.luxCDE. We have used pBAD.luxABG plasmid DNA (BBa_K1725340) for the first transformation and then transformed resulting cells with R0011N.luxCDE library which was first ligated into PSB343 (plasmid with kanamycin resistance). In terms of ribosome binding sites, we aimed to have the same type of luxABG in all cells and different type of luxCDE in each cell in order to ensure that any differences in bioluminescence arise from ribosome binding sites in the luxCDE genes. Transformed cells were grown on ara+ plate over night at 37°C and kept for an hour at room temperature before photographed (Figure 10A). Once again, we observed a range of bioluminescent colonies, and this time light output was seen by naked eye: we were able to see single colonies emitting different amounts of light.
Figure 10
Results
- Cell-cell comunication - Decanal experiments - Spectrum experiments and comparison to Cambridge operonReferences