Team:Edinburgh UG/Results
Results
Achievements
- We have characterised various methods of maxicell production, to optimise their manufacture for synthetic biology
- The active time frame of maxicells was quantified, to determine their potential “lifespan”
- We have created parts which facilitate maxicell production in any E. coli strain of your choosing
- We have introduced a functional biosensor into the maxicell chassis, which has shown that maxicells have the potential for gene expression for at least 18 hours after their production
- Various methods of preventing horizontal gene transfer were explored, to increase the security of maxicells as a chassis designed for environmental release
- Triclosan resistance was introduced into a standard backbone as an alternative to antibiotic selection, which would prevent the unwanted release of antibiotic resistance genes
Evaluating and Optimising Maxicell Protocols
Three methods of maxicell production were evaluated to identify the most efficient process. Each method causes a double-strand break in the chromosome, leading to its degradation by native exonucleases. After maxicell induction, all of these methods involved treatment by cycloserine to kill the non-maxicells. Cycloserine is an antibiotic that targets cell wall synthesis, thereby killing any actively dividing cells. The level of maxicell production was determined by fluorescence microscopy of samples stained with DAPI; a fluorescent stain that binds to double stranded DNA.
UV Production Method
We first started by looking at the Escherichia coli strains DH5-alpha (recA-, uvrA+) and CSR603 (recA-, uvrA->. It was found that when attempting maxicell formation in strains with functional uvrA, such as DH5-aplha, all stages post-UV exposure until the end of a 15-18 hour incubation must be carried out in complete darkness, to prevent photoreactivation by the UVR system. We also found that while CSR603 is a more efficient maxicell producer than DH5-alpha, it grows slower and the cells are percentage shorter in length and diameter than DH5-a. We surmised that this was because the cells are, in essence, sicker due to the mutations in DNA repair systems.
Next, we used the E. coli strain MC4100 to produce a recA, uvrA knockdown, as this strain was recommended in literature for maxicell production. Through utilisation of RNA interference by MicC sRNAs, we created a recA, uvrA knockdown construct. The objective was twofold: to develop a method of maxicell production which could be theoretically applied to any strain of E. coli; and to create a healthier maxicell producing strain - after it was observed that CSR603 has impaired growth due to its sickness. Both of these objectives were successful. For the knockdown strain, we found that the colony forming units per mL culture 18 hours post UV-exposure was vastly lower than that of MG1655 (fully functioning recA and uvrA), and only slightly higher than CSR603 (fully non-functioning recA and uvrA). Furthermore, MC4100 growth rate in LB media and M9 glucose media is vastly higher than that of CSR603. When cells are cultured in M9 glucose media, there is no significant difference in CFU/mL 18 hours post-UV exposure when cells are exposed to light or kept in complete darkness. When cells are cultured in LB media, CFU/mL is significantly higher if the post-UV culture is exposed to light.
Palindromic Cleavage Method
Another method of maxicell induction that was investigated involved using a strain of E. coli, DL3355, which contains a palindromic site in the chromosome that forms a hairpin loop. This hairpin loop can be cut by an sbcCD exonuclease that was introduced to the chromosome under control of an arabinose-sensitive promoter. This causes a double strand break in the chromosome. leading to digestion by native exonucleases. However, this method was found to be largely unsuccessful: a spot test of E. coli DL3355 with the sbcCD exonuclease gene under an arabinose inducible promoter inserted into the chromosome showed that there was no decrease in colony forming units after sbcCD induction. It was surmised that this was due to a high rate of mutation in this strain, as the palindromic sequence is a burden on the cell there is an inherent rate of loss of the palindrome site of 1/10000 per genome per generation. The rate of strain reversion is therefore very high, so this method of maxicell production is inefficient and not recommended.
Homing Endonuclease Method
The last method we looked at centres around E. coli DL2524, a strain that contains in its chromosome an 18 base pair recognition site for the homing endonuclease Isce-I, and the Isce-I gene under control of an arabinose-sensitive promoter. Maxicell production by this strain was initiated by culturing in arabinose, which activates Isce-I expression, leading to a double-strand break in the chromosome at the Isce-I recognition site. This method was the most successful at maxicell production, as seen by fluorescence microscopy after DAPI staining.
Quantifying Maxicell Timeframe
ATP Quantification
Protein Retention
Next, we decided to investigate the level of protein degradation in our maxicells, to find out whether or not all the cellular machinery remains intact over long periods of time. To do so, we took samples of maxicells stored 4°C for 0 h and 24 h, and at 25°C & 37°C for 0 h, 24 h and 48 h. We then ran these samples through an SDS-PAGE gel, the results of which can be seen in figure (______) below. These results show that there is no major decrease in protein concentration within maxicells over a period of 48 h, not even at 37°C, suggesting that the level of protein degradation is minimal. Although we cannot determine absolute protein concentrations between the samples due to potential differences in sample preparation, we can see that they are not orders of magnitude different. It can therefore be inferred that protein degradation within maxicells is not a significant factor for their functionality over long periods of time.
Functional Gene Expression
Finally, CSR603 UV-induced maxicells were used to investigate the ability of maxicells to transcribe and translate a chosen gene, in this case mCherry under an inducible arabinose promoter. These maxicells were induced by arabinose to produce mCherry 18 hours after UV exposure. Fluorescence by mCherry was observed after arabinose treatment, and for at least 24 hours further. The proportion of cells fluorescing 24 hours after mCherry induction significantly decreases in samples induced 19 hours after UV exposure, and a maximum of 6% of cells fluorescing was seen in samples induced 25 hours post-UV exposure. In contrast,it was found that cells with intact chromosomes fluoresce only faintly after arabinose treatment, and mCherry is not visible to the naked eye in colonies cultured on LB+arabinose plates. It can be surmised, therefore, that maxicells can be induced to produce a gene product after their production, and can act as a biosensor: for arabinose in this case.
Unfortunately, due to difficulties in transformation of CSR603 we were unable to produce a functional ars-mCherry construct in this strain. This made it impossible to test the ability of maxicells to act as a biosensor for arsenic.
The Triple Lock System
Colicin Kill Switch
Semantic Containment
Alternative Selection
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