Difference between revisions of "Team:Glasgow/Project/Overview/Bioluminesence"

Introduction

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 2011, 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.

Our Aims

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.



Figure 1 – Biobrick K325909 in pSB1C3 showing Gene Order of luxCDABEG. This biobrick constructed by the Cambridge 2010 iGEM team contains the native operon isolated directly from V. fischeri.

Getting Started

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 - Lux A
K1725207luxB
K1725208luxC
K1725209luxD
K1725210luxE
K1725211luxG

Creating LuxA-LuxB-LuxG

(Top 10 cells were use for all transformations using these genes) We made an attempt to insert Lux B downstream of Lux A by using the biobrick assembly method, and then transformed our ligations into Top10 cells. The first transformation plates showed no growth, so we decided to repeat the transformation with new competent cells. However, there was still not as much growth as expected, suggesting an issue with the ligation. To overcome this, we decided to ligate the genes together using a different method. Lux A would be inserted upstream of Lux using different enzymes from the ‘standard’ biobrick assembly; insert cut with AlwN1 and Spe1 (Lux A), and vector cut with AlwN1 and Xba1(Lux B ). AlwN1 sticky ends were able to ligate together, and the ends of Xba1 were able to ligate to the beginning of the Spe1 cut site. This method was very successful and lots of colonies were visable on the transformation plates! A miniprep DNA sample run on a gel confimed that the two genes had been successfully ligated together.
•K1725212 – Lux A & Lux B

Arabinose promoter – pBAD - was inserted into K1725212 upstream of Lux A to test and prove that Lux A and Lux B when switched on could cause cells to produce bioluminescence (after decanal was added) (Figure 4). This was done again using AlwN1 and Spe1 to cut the insert, and AlwN1 and Xba1 to cut the vector. 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 Lux A and B were both switched off when the promoter is not induced.
•K1725214 – pBad, Lux A & Lux B

The next step was to insert Lux G into the LuxA/B plasmid and the pBAD-LuxA/B plasmid, downstream of Lux B using the biobrick assembly method. However, there an issue with this ligation, no colonies were produced after two separate attempts (Cell control on both attempts grew as expected) using Top10 cells. LuxA & LuxB together were much too large to switch the ligation as before and insert them upstream of LuxG. Because of this, we arrived at the conclusion that LuxG could have been slighty expressed with no need for a promoter, and the gene product was toxic to the cells when Lux A and Lux B were not being expressed. Therefore, baring in mind our time limit, we decided to omit Luc G from any further ligations, and we decided to make our final ‘product’ without Lux G.

Creating Lux C, Lux D & Lux E

When putting together Lux C & Lux D, it was decided to insert Lux C upstream of Lux D in the same was as for Lux A & B, again using AlwN1 and Spe1 to cut the insert, and AlwN1 and Xba1 to cut the vector was cut out of pSB1C3. The ligation was successfully transformed into Top10 and this gave another us another new biobrick.
•K1725213 – Lux C & Lux D

The next step was to insert a promoter upstream of LuxC/D , as we had planned some experiments to measure the levels of decanal produced Lux C, Lux D and Lux E were expressed. We decided to use the R0011N- IPTG promoter, which was inserted upstream of Lux C/D and transformed into Top10 cells. However, no growth occurred on plates. After some discussion, we realised that this ligation was not ‘good’ for the Top10 cells, as the promoter will always be induced in the strain. To overcome this, we decided to try and transform DH5a and DS941.Z1 cells with the original LuxC/D plasmid, as in these strains the promoter would not be automatically induced once ligated to LuxC/D, and therefore the genes would not unintentionally be expressed. The transformation was done before any more attempts at inserting the R0011N promoter, and we mini-prepped the LuxC/D vector for the new strains to repeat the ligation with this new DNA. pSB1C3-LuxE was also transformed into DS941.Z1 to prepare for the proceeding steps.

A second attempt at inserting the promoter upstream of LuxC/D was completed using the AlwN1-Xba1-Spe1 method, and ligations were transformed into DH5a cells. At the same time, a separate ligation was set up, where LuxE was inserted downstream of LuxC/D and also transformed into DH5a cells. This resulted in another two biobricks;
•K1725222 – Lux C, Lux D, Lux E
•K1725215 – R0011N, Lux C, Lux D

A final ligation was set up to ligate Lux E and R0011N- Lux C/D. Lux E was inserted downstream of Lux D.
•K1725219 – R0011N-LuxC, LuxD, LuxE

Creating Final Products

For the final product all that was left to do was join together LuxA/LuxB to LuxC/LuxD/LuxE, bearing in mind Lux G was intended to have been included, but proved too difficult. Lux C/D/E were cut out of pSB1C3 and inserted downstream of LuxA/B and also pBadLuxA/B so that we had another biobrick to add to our growing list.
•K1725223 - LuxA/B/C/D/E (Figure 5)
•K1725224 - pBAD LuxA/B/C/D/E

A gel was creating using restriction digests of all biobricks being submitted, where insert was cut out of vector with EcoR1 and Pst1 each time (Figure 6).

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