Team:UiOslo Norway/Existing Part
Contributing to the bigger picture
Existing part
Part code: BBa_K274120
Source:Pantoea ananatis
Figure 1: The composition of BBa_K274120 with its promoter, the genes with individual RBS.As we were producing lycopene by sequential cloning of crtE, crtB and crtI from Deinococcus radiodurans into Escherichia coli, we looked for other similar parts in the registry. However, not all of them were well characterized, which gives us a great opportunity to do so. We settled on a part similar to our own to be able to make the best characterization and comparison to our own part. There were many similarities between our composite part and BBa_K274120, both use the same arabinose inducible promoter, both catalyze the same reactions and use the same genes.
The difference between our pBAD-crtEBI and the already existing part is that BBa_K274120 has individual ribosomal binding sites (RBS) between each enzyme within their composite part. In comparison, our part has a single RBS followed by all three genes in a single open reading frame.
Characterization
Both constructs are cloned into pBAD vector and transformed into E. coli BL21 for characterization. E. coli was induced with 2% arabinose, resulting in the red coloration on pellet, confirming that lycopene is present. Figure 2 compares uninduced and induced E. coli, both with empty pBAD vector as well as the construct. By looking at the picture you can clearly see a red colour on the pellet from the induced E. coli containing the construct, while both uninduced samples and the empty vector have an off-white/pink colour.
Figure 2: From left to right: Uninduced E.coli with empty expression vector, induced E. coli with empty expression vector, uninduced E. coli with BBa_K274120, induced E. coli with BBa_K274120
Figure 3 presents our final composite part in the same way as the characterized part. From the figure it is possible to see colour difference between the induced and uninduced lycopene producing cells, as well as between the controls without either construct.
Figure 3: From left to right: Uninduced E. coli with empty expression vector, induced E. coli with empty expression vector, induced E. coli with crtEBI expression vector, uninduced E.coli with crtEBI expression vector
By comparing the red colour of the pellets in figure 2 and 3, we can conclude that the induced culture from BBa_K274120 produced far more lycopene than BBa_K2971004.
Sequencing of BBa_K2971004 indicated that it had a frameshift mutation resulting in the loss of the stop codon and potentially explaining our results. By removing the stop codon our construct will be longer, which may introduce different protein foldings and provide other properties.
For further characterization, we tried to get a growth curve for BBa_K274120 using a microplate reader. At the same time we wanted to measure lycopene production over 48 hours after induction. We observed that irrespective of the insert, cells had slower growth when induced compared to uninduced cells as shown in figure 4. The readings at 472nm seemed to be affected by cell mass and bacterial products.
Figure 4: Comparison of growth between BBa_K274120 (HQ) and pBAD
In addition to the growth curve, we tried to calculate the lycopene production after 48 hours by extracting it with acetone. We hoped to have no interference from the cell mass in this experiment. However, the results we got were inconsistent, and we believe that the reason is failed cell lysis. To protect the instrument, we had to dilute the acetone. Because of this we think that the cell lysis failed. Other methods of cell lysis may perform better.
We tried direct detection of lycopene within the lysate, without good results. Because of these results we tried thin layer chromatography (TLC). Before starting we separated lycopene from the cells by extraction with benzene. Results are shown in figure 5 with both visible and UV light. The red spots on the paper confirms that lycopene is produced.
Figure 5: TLC result in visible (left) and in UV (right). From left to right: induced BBa_K274120, uninduced BBa_K2971004, induced BBa_K2971004.
We can visualize the production of lycopene but is difficult to quantify it. For quantification more specialized techniques such as High Performance Liquid Chromatography, Mass Spectrometry or others could be used.