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

We as a team were fascinating by the glowing pictures taken from Team Cambridge 2011, but we wanted to make our cells grow even brighter! To do this, we aimed to recreate 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 then progressing to using a combination of random Ribosome Binding Sites (RBS) for each gene that resulted in the brightest colonies (HYPERLINK TO RBS STUFF???)

This sub-part to the project works alongside the UVA-sensor (HYPERLINK???) and Repressors(HYPERLINK???) to make up our complete project.

Getting Started

To begin, we created primers for the individual Lux Genes using biobrick K325909 as a template. All primers were made to contain the B0032 RBS. Our primers were used to carry out PCR aiming to obtain individual Lux Genes (Figure 3). Once each of the genes was successfully PCR’d, they were ligated individually into pSB1C3 vectors and Transformed individually into Top10. These ligations went on to become our first biobricks submitted for the Bioluminescence section. ( K1725206-K1725211)

•K1725206 – Lux A in pSB1C3
•K1725207 – Lux B in pSB1C3
•K1725208 – Lux C in pSB1C3
•K1725209 – Lux D in pSB1C3
•K1725210 – Lux E in pSB1C3
•K1725211 – Lux G in pSB1C3

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

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Protocols

UVA

Terminator

Repressors