dCas9

Once we had decided on the dCas9 system, we used benching to design gRNA inserts to target eKatg, mScar and a non-targeting control. Plasmids were designed to minimise off target binding, whilst maintaining the protospacer adjacent motif. We added BsaI sites to the gRNA inserts, to make them compatible with the BsaI plasmid insertion.

The plasmid, and gRNA sequences were then ordered from IDT. The gRNA primers were ordered as single strands to save costs.

When the gRNA strands arrived, they were hydrated and oligo annealed by rapidly heating, and slowly cooling, the strands in the presence of an annealing buffer.

Following annealing, the gRNAs were inserted into the pdCas9 plasmids using a BsaI golden gate reaction, which ran over night.

Once the BsaI reaction was complete, the plasmids were transformed into host (ecoli strain) via electrocution. The cells were allowed to divide before being plated on chloramphenicol infused agar.

The agar plates were observed for growth, and colonies were taken and grown in LB broth with chloramphenicol. The pdCas9 plasmid contained a mRFP dropout, and as such, successful BsaI reactions were identified from the absence of red.

The plasmids contained chloramphenicol resistance genes, allowing unsuccessful transformations to be removed.

The selected colonies were then incubated overnight at 37°C.

gRNA plasmids were isolated from starter culture following the Isolate II Meridian low copy number protocol. Isolated plasmids were then digested with BamHI-HF and SacII, using the following conditions:

37°C for 4 hours, followed by 80°C for 20 minutes.

The digested plasmid solution was then separated by gel electrophoresis to confirm that the gRNA inserted into the plasmid backbone appropriately.

Having confirmed the successful insertion of gRNA into the plasmid backbone, we transformed E. coli K-12 with the plasmids and grew them on chloramphenicol infused agar.

We attempted to select colonies, however the transformation was not successful

E. coli K-12 was transformed with appropriate plasmids containing gRNA.

The agar plates were once again observed for growth. The plates contained ecoli with the purified pdcas9 plasmid, and either a rhamnose inducible mScar plasmid, or a constitutive mScar plasmid. The presence of the pdcas9 plasmids was screened using chloramphenicol resistance. Whilst the mScar plasmids were screened via colour, and carbenicillin, and spectinomycin, respectively.

To confirm that the dCas9 knockdown system worked as reported by Bradley (2021), we repeated their experiment, following similar concentrations of anhydrotetracycline and arabinose. Our supervisor, Andrew George, noted that the reported optimal concentration of arabinose used by Bradley (2021) was significantly lower than what was traditionally used by others. And thus, we introduced an additional trial for a higher arabinose concentration.

The fluorescence of the cell suspensions were corrected for optical density and compared. Levels of activity reduction did not match our guide paper. Our supervisor explained that they usually use higher arabinose concentrations then that which was listed in the paper, so we intend to attempt to survey if different levels work better.

This first test result was disheartening; however, we believe that we can still make it work of we optimization the promoters. This does become difficult, as the pdCas9 plasmid has two promoters, anhydrotetracycline for the dCas9 protein, and L-arabinose for the gRNA and its associated scaffold.

The fluorescence reading showed that the higher L-arabinose concentration resulted in a slightly higher knockdown of mScar. We decided to repeat the mScar knockdown experiment, however we attempted to use different concentrations of L-arabinose.

Once again, we noticed that the CRISPRi system did not result in the 40-fold reduction seen by Bradley (2021). However, the higher arabinose concentration did result in a greater knockdown of mScar.

Altered L-arabinose and anhydrotetracycline (aTc) concentrations to optimize CRISPRi function. Interestingly, previous results didn’t replicate showing that lower L-arabinose performed better than higher concentrations. aTc concentration had no effect on CRISPRi repression.

New pdCas9 starter cultures were developed from the MiniPrep stock.

Used yesterday's starter cultures to generate 4 reaction systems:

• pdCas9 E. coli KatG + Ara + Atc
• pdCas9 E. coli KatG + Ara
• pdCas9 mScar3 + Ara + Atc
• pdCas9 mScar3 + Ara

The solutions were incubated for 2 hours, to allow them to reach 0.80 OD, at which point, H2O2 was added to the solutions. The OD was then monitored to observe for changes in the growth patterns. No changes were observed.

The previous results have all been lackluster, so we set out to sequence our gRNA segments, to ensure that they are effectively targeting their respective proteins. We designed our PCR promoters, and ran our PCR with dideoxy nucleotides to enable us to sanger sequence the gRNAs.

Following PCR, we submitted our samples to the ANU sanger sequencing laboratory.

The results indicated that the gRNAs were perfect. This was the expected result; however, we were hopeful that the reason for our lackluster results was incorrect targeting.

We brainstormed potentially ordering new gRNAs which target the promoters of our target proteins, however this would require designing a new CRISPR system, as the pdCas9 system would not be able to target our particular protein promoters.

We did not have time to order new plasmids, and run our desired experiments, so we cut our losses and discontinued our CRISPR portion of the project.

Golden Gate Assembly and Initial Transformations

Performed Golden Gate reactions for constructs 1-5 and 9-11, and transformed them into E. coli DL21 cells. The transformed cells were then plated on agar containing ampicillin to select for those that successfully incorporated the plasmids.

Cells expressing plasmids 9-11 failed to grow, which may have been due to a missed component in the master mix created for these reactions. We replated these failed transformations of constructs 9-11 using our existing Golden Gate mixtures.

Picked colonies for constructs 1-5 and 9-11 from our plates. Each selected colony was inoculated into LB broth with ampicillin to establish cultures for further analysis.

Conducted miniprep of constructs 1-5 and 9-11.

Level 2 Assembly and Transformations

Set up level 2 Golden Gate reactions for constructs 6-8. These constructs are designed to combine elements from constructs 1-5 to create complex biosensor systems, with RFP under the control of our 3 ROS inducible promoters.

Transformed reactions for constructs 6-8 into E. coli DL21 cells using electroporation. The transformed cells were then plated on agar containing spectinomycin to select for those that successfully incorporated the plasmids.

Picked cultures for constructs 6-8 and incubated them overnight to allow for adequate growth and plasmid replication.

Miniprepped cultures for constructs 6-8 using a series of centrifugation and buffer steps.

Combining Constructs and Verification through Digestion

Performed transformations of constructs 6-8 with constructs 9-11 to generate more integrated systems. Plated these cells on plates containing both spectinomycin and ampicillin, to select for both the catalase and biosensor plasmids. In these cells, catalase expression is inducible by rhamnose, while the ROS-inducible promoters regulate RFP.

Conducted overnight digests using NdeI restriction enzymes. This process involved mixing plasmid DNA with buffer, water, and the NdeI enzyme, then incubating overnight.

Hydrogen Peroxide Experiments

We found that colonies containing plasmid 7 (specifically constructs 7+9, 7+10 and 7+11) exhibited extensive red fluorescence. This was unexpected given the absence of added ROS, as plasmid 7 contains the ROS-sensitive promoter Trxcp which should not lead to significant expression under these conditions. This suggests that the promoter may be leaky. Consequently, we decided to exclude these constructs from further experiments. Instead we initiated starter cultures for the following plasmid combinations: 6+9, 6+10, 6+11, 8+9, 8+10, and 8+11. Each culture was transferred to a 50 mL Falcon tube containing LB broth supplemented with spectinomycin and ampicillin.

Following our experimental procedures, we incubated cells containing constructs 6+9, 6+10, 6+11, 7+9, 7+10, and 7+11 for 6 hours with varying concentrations of hydrogen peroxide. For these experiments we prepared 5 concentrations for each plasmid construct: 0mM, 5mM, 10mM, 20mM and 30mM. After 6 hours, we measured optical density (OD) at 600nm and fluorescence with excitation at 570nm and emission at 610nm to assess the biosensor response to oxidative stress and the resilience of our cells.

Following our initial incubation, we realized that we had omitted ampicillin when preparing the cells which compromised selection for the catalase plasmids. To address this, we repeated the incubation with hydrogen peroxide, this time allowing the cells to incubate overnight in a shaking incubator.

Remeasured absorbance and fluorescence for all cultures. These results showed improved responses compared to the previous tests, though the cells still struggled to survive even the lowest hydrogen peroxide concentrations. In light of this, we decided to lower both the concentrations and the incubation times for subsequent experiments.

ncubated cells for the third time with hydrogen peroxide, this time using 0mM, 2.5mM, 5mM, 10mM and 20mM H2O2. Measurements of absorption and fluorescence were taken at 1h, 3h and 4.5h intervals to evaluate the time-dependent effects of hydrogen peroxide on cell viability and biosensor performance.

We found that our results still did not adequately demonstrate biosensor accuracy or cell tolerance to oxidative stress. Instead, we decided to follow the alternative approach detailed in our experimental procedures to incubate cells with hydrogen peroxide, aiming to optimize cell growth for better survival and more reliable biosensor readings.

References