- BBa_K1725350: I0500-RBS-luxA-RBS-luxB-RBS-luxG
- BBa_K1725351: K1725080-RBS-luxC-RBS-luxD-RBS-luxE
- BBa_K1725352: I0500-RBS-luxA-RBS-luxB-RBS-luxG-K1725080-RBS-luxC-RBS-luxD-RBS-luxE
Difference between revisions of "Team:Glasgow/Description"
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<img src="https://static.igem.org/mediawiki/2015/0/04/Figure_2_Sequencing.jpeg" style="width: 500px; " /> | <img src="https://static.igem.org/mediawiki/2015/0/04/Figure_2_Sequencing.jpeg" style="width: 500px; " /> | ||
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− | <figcaption><b>Figure 2. Sequencing results of <i>luxA</i> amplified with a primer involving randomised RBS B0032 sequence. </b> 4 peaks at the same position are observed where sequence was randomised to N and 2 peaks are observed | + | <figcaption><b>Figure 2. Sequencing results of <i>luxA</i> amplified with a primer involving randomised RBS B0032 sequence. </b> 4 peaks at the same position are observed where sequence was randomised to N and 2 peaks are observed where nucleotide location was randomised to R.</figcaption> |
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<img src="https://static.igem.org/mediawiki/2015/2/24/Figure_5_LuxAB_coloniesAJ.png" style="width: 700px; " /> | <img src="https://static.igem.org/mediawiki/2015/2/24/Figure_5_LuxAB_coloniesAJ.png" style="width: 700px; " /> | ||
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− | <figcaption>Figure 4</figcaption> | + | <figcaption><b>Figure 4. pBAD.luxAB colonies A-J. </b> Initial transformation plate with marked locations where colonies A-J where picked (left)and short streaks of colonies A-J. Yellow: bright colonies, Orange: medium-brightness colonies, RED: Dim colonies (as appears on the transformation plate) </figcaption> |
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<div class="text-center"> | <div class="text-center"> | ||
− | <img src="https://static.igem.org/mediawiki/2015/c/c0/Figure_7.png" style="width: | + | <img src="https://static.igem.org/mediawiki/2015/c/c0/Figure_7.png" style="width: 900px; " /> |
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− | <figcaption>Figure 6</figcaption> | + | <figcaption><b>Figure 6. Pictures of pBAD.luxABG colonies streaked on ara+ and ara- plates in white light and in the dark. </b> Plate on the left side contains L-arabinose, plate on the right is a control.</figcaption> |
</br> | </br> | ||
Additionally, in order to compare bioluminescence between cells where <i>luxG</i> is expressed and in cells where it is not, we also streaked previously described 12 pBAD.<i>luxAB</i> colonies and 12 brightest pBAD.<i>luxABG</i> colonies on the same ara+ plate. As can be seen from the Figure 7, most of the pBAD.<i>luxABG</i> colonies appear to exhibit brighter luminescence than the pBAD.<i>luxAB</i> colonies which is what we expect to see. | Additionally, in order to compare bioluminescence between cells where <i>luxG</i> is expressed and in cells where it is not, we also streaked previously described 12 pBAD.<i>luxAB</i> colonies and 12 brightest pBAD.<i>luxABG</i> colonies on the same ara+ plate. As can be seen from the Figure 7, most of the pBAD.<i>luxABG</i> colonies appear to exhibit brighter luminescence than the pBAD.<i>luxAB</i> colonies which is what we expect to see. | ||
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<img src="https://static.igem.org/mediawiki/2015/b/b4/Figure_8.png" style="width: 300px; " /> | <img src="https://static.igem.org/mediawiki/2015/b/b4/Figure_8.png" style="width: 300px; " /> | ||
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− | <figcaption>Figure 7</figcaption> | + | <figcaption><b>Figure 7. Picture of pBAD.luxAB and pBAD.luxABG colonies streaked on ara+ plate taken in the dark. </b> Left bottom corner: 12 pBAD.luxAB colonies; right bottom corner 12 'bright' pBAD.luxABG colonies; top and middle: 136 random pBAD.luxABG colonies. </figcaption> |
We have picked up the brightest pBAD.<i>luxABG</i> colony, determined RBS for each gene by sequencing and submitted the construct as the biobrick BBa_K17252340. | We have picked up the brightest pBAD.<i>luxABG</i> colony, determined RBS for each gene by sequencing and submitted the construct as the biobrick BBa_K17252340. | ||
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<img src="https://static.igem.org/mediawiki/2015/7/7d/Figure_9.png" style="width: 800px; " /> | <img src="https://static.igem.org/mediawiki/2015/7/7d/Figure_9.png" style="width: 800px; " /> | ||
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− | <figcaption>Figure 8</figcaption> | + | <figcaption><b>Figure 8. Testing 2-cell communication system. </b> Flasks with the same pBAD.luxABG (BBa_K17252340)and different R0011N.luxCDE colony in each flask photographed in white light and in the dark. </figcaption> |
</br> | </br> | ||
The second strategy that we have developed for testing our RBS library in <i>luxCDE</i> assembly involved generating <i>E. coli</i> strain with two plasmids: PSB1C3 with pBAD.<i>luxABG</i> and PSB343 with R0011N.<i>luxCDE</i>. We have used pBAD.<i>luxABG</i> plasmid DNA (BBa_K1725340) for the first transformation and then transformed resulting cells with R0011N.<i>luxCDE</i> 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 <i>luxABG</i> in all cells and different type of <i>luxCDE</i> in each cell in order to ensure that any differences in bioluminescence arise from ribosome binding sites in the <i>luxCDE</i> genes. Transformed cells were grown on ara+ plate over night at 37°C and kept for an hour at room temperature before photographed (Figure 9). 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. | The second strategy that we have developed for testing our RBS library in <i>luxCDE</i> assembly involved generating <i>E. coli</i> strain with two plasmids: PSB1C3 with pBAD.<i>luxABG</i> and PSB343 with R0011N.<i>luxCDE</i>. We have used pBAD.<i>luxABG</i> plasmid DNA (BBa_K1725340) for the first transformation and then transformed resulting cells with R0011N.<i>luxCDE</i> 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 <i>luxABG</i> in all cells and different type of <i>luxCDE</i> in each cell in order to ensure that any differences in bioluminescence arise from ribosome binding sites in the <i>luxCDE</i> genes. Transformed cells were grown on ara+ plate over night at 37°C and kept for an hour at room temperature before photographed (Figure 9). 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. | ||
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<img src="https://static.igem.org/mediawiki/2015/c/cd/Figure_10_updated_night_sky.png" style="width: 700px; " /> | <img src="https://static.igem.org/mediawiki/2015/c/cd/Figure_10_updated_night_sky.png" style="width: 700px; " /> | ||
</div> | </div> | ||
− | <figcaption>Figure 9</figcaption> | + | <figcaption><b>Figure 9. Plates of cells transformed with pBAD.luxABG (BBa_K17252340) and RBS library of R0011N.luxCDE in PSB3K3. </b> Left: Picture taken after 1hr at room temperature; right: picture taken after 5hrs at the room temperature </figcaption> |
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<img src="https://static.igem.org/mediawiki/2015/1/19/Figure_11.png" style="width: 900px; " /> | <img src="https://static.igem.org/mediawiki/2015/1/19/Figure_11.png" style="width: 900px; " /> | ||
</div> | </div> | ||
− | <figcaption>Figure 10</figcaption> | + | <figcaption><b>Figure 10. Plates with pBAD.luxAB colonies exposed to decanal.</b> Left: plates without the lids; right: lids with decanal on the plates.</figcaption> |
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<img src="https://static.igem.org/mediawiki/2015/c/c4/Figure_11_Flasks.png" style="width: 700px; " /> | <img src="https://static.igem.org/mediawiki/2015/c/c4/Figure_11_Flasks.png" style="width: 700px; " /> | ||
</div> | </div> | ||
− | <figcaption>Figure 11</figcaption> | + | <figcaption><b>Figure 11. Comparison to luxCDABEG (BBa_K325909)</b> Left: flask with BBa_K1725352 colony (GlasGlow); right: flask with BBa_K325909 colony (E. glowli) </figcaption> |
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<img src="https://static.igem.org/mediawiki/2015/0/0e/Figure_12.png" style="width: 800px; " /> | <img src="https://static.igem.org/mediawiki/2015/0/0e/Figure_12.png" style="width: 800px; " /> | ||
</div> | </div> | ||
− | <figcaption>Figure 12</figcaption> | + | <figcaption><b>Figure 12</b></figcaption> |
</br> | </br> | ||
From the calculations given by the RBS library calculator, we can clearly see that almost all of our RBS sequences (except RBS of <i>luxB</i> and <i>luxC</i>) from our brightest strain are estimated to be more efficient than B0032 in translation initiation. Therefore, we conclude that we have increased bioluminescence in <i>E. coli</i> by successfully adjusting ribosome binding sites of <i>lux</i> genes to the translation mechanism in <i>E. coli</i>. | From the calculations given by the RBS library calculator, we can clearly see that almost all of our RBS sequences (except RBS of <i>luxB</i> and <i>luxC</i>) from our brightest strain are estimated to be more efficient than B0032 in translation initiation. Therefore, we conclude that we have increased bioluminescence in <i>E. coli</i> by successfully adjusting ribosome binding sites of <i>lux</i> genes to the translation mechanism in <i>E. coli</i>. |
Revision as of 21:55, 17 September 2015
RBS Library
Home > Project > 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
Biobricks
Composite Parts
Basic Parts: RBS library
- BBa_K1725301
- BBa_K1725302
- BBa_K1725303
- BBa_K1725304
- BBa_K1725305
- BBa_K1725306
- BBa_K1725307
- BBa_K1725308
- BBa_K1725309
- BBa_K1725310
- BBa_K1725311
- BBa_K1725312
- BBa_K1725313
- BBa_K1725314
- BBa_K1725315
- BBa_K1725316
- BBa_K1725317
- BBa_K1725318
- BBa_K1725319
- BBa_K1725320
- BBa_K1725321
- BBa_K1725322
- BBa_K1725323
- BBa_K1725324
- BBa_K1725325
- BBa_K1725326
- BBa_K1725327
- BBa_K1725328
- BBa_K1725329
- BBa_K1725330
- BBa_K1725331
- BBa_K1725332
Introduction
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
Testing pBAD.luxAB
Mr. Bright: luxG
Story about luxCDE
Results
Decanal
Cell communication
Comparison to luxCDABEG (BBa_K325909)
RBS optimisation
References