Team:CKWA-China/Proof Of Concept
Proof Of Concept
Proof Of Concept
We were able to complete experiments and made measurement on our parts. We designed, built and measured a new part. The basic part is rLon (E.coli)-6xHis (http://parts.igem.org/Part:BBa_K3825000), which is the coding sequence of Lon protease obtained from E. coli sources. According to the reports in literatures, Lon protease decomposes C-Myc protein commonly found in cancer cells. Because of this potential, the protease is planned to be used to make medicine to suppress cancer recurrence. The part will be expressed in composition with LacI and LacO components together with a T7 promoter, and this composite parts is documented as T7-LacO-rLon (E.coli)-6xHis (http://parts.igem.org/Part:BBa_K3825001).
1. Vector design
We designed to use LacI and LacO together with T7 promoter to express this Lon protease in a commonly used plasmid pet28a(+).
For the measurement, E. coli Rosetta containing the designed plasmid was incubated at 37C overnight with 1mM IPTG added. Then the cells are lysed by 200 μL 4% SDS for 10 minutes in room temperature, then 10 min at 95C. Then loading buffer is added. The SDS-PAGE was performed with a 12% precast polyacrylamide gel. Coomassie bright blue stain is used to show the bands.
Figure.1. The design of part rLon (E.coli)-6xHis
2. Protein optimization
For the measurement, E. coli Rosetta containing the designed plasmid was incubated at 37C overnight with 1mM IPTG added. Then the cells are lysed by 200 μL 4% SDS for 10 minutes in room temperature, then 10 min at 95C. Then loading buffer is added. The SDS-PAGE was carried with a 12% precast polyacrylamide gel. Coomassie bright blue stain is used to show the bands.
With these induction conditions, in our first test we failed to observe a clear band of Lon protein. The bacteria culture was also relatively clear. It is possible that Lon protease has been decomposing the proteins needed for E. coli growth. Because the bacteria growth was suppressed by the Lon protease, its own production was low.
Figure.2. Bacteria Culture overnight and the SDS-PAGE results with/without iPTG
When there is LacI and there is LacO before the Lon gene sequence, IPTG can be used to control the expression. The control can be tuned by the concentration and timing of induction. We then changed our condition: the bacteria were cultured on LB plate overnight at 37C, and then pick a single colony and incubate in LB medium until the OD600 reached 0.6-0.8. At this time, IPTG was added at 0.5 mM, and the medium was again incubated at 37C for another 4 hours. This time the SDS-PAGE results showed remarkable expression of the Lon protease. The results confirmed the capability of this part, and also showed for better protein expression and better bacteria growth, the timing for induction should also be considered and experimented.
Figure.3. Expression of Lon protease confirmed in SDS-PAGE.
Lon protease may have an impact on bacterial growth, and it is necessary to control appropriate induction reagents and culture conditions for better expression. We have initially found suitable conditions, and we can continue to optimize on this basis.
Figure 4. (A) Expression of Lon protease under different conditions of IPTG concentration, OD600 at induction and different E. coli strain. (B) the SDS-PAGE results of the corresponding sample. All sample were induced and then incubated at 37C.
First we consider to optimize for the concentration of IPTG used for induction. Lane 4, 5 and 6 used the same E. coli strain, namely BL21(DE3), but they used different concentration of IPTG induction (Lane 4: 0.5 mM, Lane 5: 1.0 mM, Lane 6: 2.0 mM). The color of the bacteria cultures look almost the same, but the sample on Lane 4 with 0.5 mM IPTG induction did not show a clear band like those in Lane 5 and Lane 6, meaning it did not express as much Lon protease. The same trend is observed for Lane 10, 11 and 12. These three lanes correspond to the samples using Rosetta (DE3) E. coli strain. Lane 10 used 0.5 mM IPTG and it did not express a remarkable band. These results suggested induction concentration of IPTG should be greater than 0.5 mM for better expression results.
Then we consider to optimize for the timing of IPTG induction. Comparing the SDS-PAGE bands for samples in BL21 Lane 1 with Lane 4 (0.5 mM), Lane 2 with Lane 5 and Lane 3 and with Lane 6. Each pair used the same concentration of IPTG induction, but the former samples were induced at OD600=0, while latter sample were induced at OD600=0.6~0.8. In all three comparison the latter samples in pairs did not express a clear band. Comparing samples in Rosetta, we would end up with the same conclusion. The results suggested induction at OD600=0.6~0.8 is a favorable condition to express Lon protease in the E. coli strains.
At last we compare between the use of different E. coli strain. The bands at same OD600 induction timing with the same induction concentration show little to no difference between the two strains, when observed with naked eyes.
In conclusion, in the future to obtain higher output, we need to use more than 0.5 mM IPTG for induction at OD600=0.6~0.8. Both BL21 and Rosetta strain would work nicely for the expression. There might be other conditions to optimize for the expression, such as the incubation temperature and incubation time. When we can fully optimize the expression and then extract the protein, we can then test the function of the protein and then determine the activity of the protein.
We used BL21(DE3) E. coli, incubated at 37° and induced with IPTG at 0.5 mM or higher at OD600=0.6~0.8, and gave the experimental material to BGI-write for our protein purification operation. The purification method of 6xHis tag was designed, and the purified Lon protein was finally obtained.
3. Future plans
Figure.4.Purified rLon protease.
We will continue to explore the conditions for optimizing the expression of Lon proteins and, according to our plan, we will also try expression in different cell systems, such as mammalian cells and several cell lines such as yeast.
Our subsequent goal is to find a cell room to perform Hela cell recovery culture experiments to verify that our production of Lon protein inhibits the C-myc gene in a manner consistent with what is documented in the literature.Due to the limitations of COVID-19, we were not able to find a cell house laboratory that would allow us to use it this year, and we hope that we will be able to perform the next experiments in the future when the epidemic is in remission.
We understand that the life cycle of a drug is extremely long and tedious: it must go through many regulated stages to ensure its safety, efficacy and quality for patients. lon protein drugs may still have very many unknowns as to whether they will make it to the clinic and to the market, but we are willing to work for it.