In this section of our project we aimed to engineer bacteria that are capable of producing bioluminescence. We were greatly inspired by the work of the Cambridge iGEM team from 2010, and used some of their research as a starting point for our own. Their project featured the Lux Operon K325909, originally from Vibrio fischeri, comprising of six lux genes that when expressed are capable of producing bioluminescence. These genes were luxC, luxD and luxE encoding three enzymes that act together to produce tetradecanal in the cell, and luxA and luxB which when expressed can use the decanal to produce luciferase and therefore bioluminescence. The final gene in the operon is luxG, which is thought to enhance the brightness of the bioluminescence, by producing reduced flavin mononucleotide (FMN) which is required for bacterial luminescence.
In 2010, the Cambridge iGEM team isolated the lux operon from V.fischeri, it was decided that our team would characterise the lux genes individually for submission to the registry. The Cambridge 2010 iGEM team used the native V. fischeri lux operon; each lux gene in the operon had its own (V. fisheri) ribosome binding site and the genes were in the order luxCDABEG (Figure 1). We aimed to reconstruct the lux operon for optimal performance using Biobrick assembly, changing the order of the lux genes from luxCDABEG (Figure 1) to luxABGCDE (Figure 2), and using a defined biobrick ribosome binding site (B0032) for each gene in the operon. Our team originally aimed to engineer a luxABGCDE operon biobrick with no promoter, so that we could drive expression from the promoter in our inverter, or future iGEM teams could add their own promoter. We also wanted to make a test operon controlled by the arabinose regulated pBAD promoter (BBa_I0500) to directly compare to K325909, and separately a construct where tetradecanal production was regulated independently from the light production enzymes LuxA, LuxB and LuxG. To do this we aimed to place luxABG under the control of the BBa_I0500 and luxCDE under the control of the IPTG-regulated pL-lac promoter, BBa_K1725080.
To begin, we created primers for the individual lux genes. All of the forward primers were made to contain the BioBrick prefix, the B0032 RBS and a BioBrick Scar preceding the ATG of the open reading frame. All of the reverse primers incorporated the BioBrick suffix sequence. Our primers were used to carry out PCR using biobrick K325909 as a template, yielding the six individual lux genes (Figure 3). Once each of the genes was successfully isolated by PCR, they were digested with EcoRI and PstI and ligated individually into the pSB1C3 plasmid backbone and transformed individually into Top10 E. coli cells. The plasmids isolated from these ligations went on to become our first biobricks submitted for the Bioluminescence section: • K1725206 – B0032.luxA • K1725207 – B0032.luxB • K1725208 – B0032.luxC • K1725209 – B0032.luxD • K1725210 – B0032.luxE • K1725211 – B0032.luxG
Creating K1725216 (luxABG)
(TOP10 cells were used for all transformations using these genes) The luxA gene was inserted upstream of luxB using AlwNI, SpeI and XbaI, different restriction enzymes from BioBrick Standard Assembly method, this method utilised the compatibility of SpeI and XbaI restriction sites (figure 4). DNA extracted from transformants from this ligation were tested by restriction digest and agarose gel electrophoresis to confirm the presence of the desired construct K1725212 (B0032,luxA, B0032, luxB). The arabinose-regulated promoter (BBa_I0500) was inserted into K1725212 upstream of luxA to demonstrate that when luxA and luxB were expressed and decanal was sprayed on the lid of the plate and allowed to diffuse across the plate, the cells produce bioluminescence (Figure 5). This was done again using AlwNI and SpeI to cut pSB1C3 (BBa_I0500), and AlwNI and XbaI to cut pSB1C3 (BBa_K1725212), creating pSB1C3 (BBa_K1725214; pBAD/araC, B0032, luxA, B0032, luxB) . Once transformed, 4 separate colonies were re-streaked onto plates containing arabinose, and the same 4 colonies re-streaked onto plates without arabinose. Non-arabinose plates were used as a control to show that luxA and luxB were both switched off when the promoter is not induced.
Creating K1725222 (luxCDE)
For assembly of luxCD, it was decided to insert luxC upstream of luxD in the same way as for LuxAB, using AlwNI and SpeI to cut pSP1C3 containing BBa¬¬_K1725208 (luxC), and AlwN1 and Xba1 to cut pSP1C3 containing BBa_K1725209 (luxD) (figure 5). The ligation was successfully transformed into TOP10.
Creating Final Products
For the final product all that was left to do was to ligate luxAB to luxCDE, bearing in mind luxG was intended to have been included, but proved too difficult. luxCDE was inserted downstream of LuxAB and also I0500luxAB so that we could compare this construct to our BioBrick of the lux operon with optimised RBS (more detail on our RBS library page): • K1725223 - luxABCDE (Figure 6) • K1725224 - I0500.luxABCDE