Dealing with a cloning problem

Cycle one

Design

We designed the new plasmid backbone BBa_K4727000, to test our CRISPR interference system in the two non model species A. baumannii and K. pneumoniae. For this reason, we decided to clone the dCas9 inside the plasmid, using the prefix and suffix sequences we inserted. We designed a straight forward cloning using the EcoRI and PstI cut sequences.

Build

Using the aforementioned restriction sites we proceed to clone the dCas sequence into the plasmid backbone and proceeded to clone the ligation product in E. coli to assess the correct insertion

Test

After cloning the so build construct in E. coli colony resulted positive to either gel screening or sequencing experiments, probably resulting only in recircularized plasmid.

Learn

After repeating the same experiment several times, never obtaining a positive outcome, and comparing the results with the cloning of RFP in the same backbone we had the opportunity to ponder over the copy number of this plasmid. Our experiments, and the literature, suggests that this plasmid backbone has an ORI for E. coli that provides a high number of copies (500-700). This information, together with the knowledge that the Cas protein if present in large quantities results in cellular toxicity, made us hypothesize that no positive colony could be detected as all the cells with the correct plasmid were killed due to the dCas toxicity.

Cycle two

Design

Given the results of the previous experiments, we decided to take advantage of the ORI for A. baumannii. For this reason we attempted to clone the same dCas expression cassette into the plasmid backbone but to transform the ligation product into A. baumannii, using our electroporation protocol.

Build

As mentioned above, we built the plasmid using the EcoRI and PstI cut sites present in the prefix and suffix sequences and proceeded to transform the ligation product in A. baumannii, using our electroporation protocol.

Test

After electroporation very few cells could be seen on the plate after overnight incubation, they were all tested for positive insertion of the dCas construct via colony PCR screening, but none resulted positive.

Learn

Electroporation utilizes a current shock to allow the entrance of DNA inside the bacterial cell; for this reason the process is really sensitive to the presence of ions inside the bacterial suspension as they allow the current to flow inside the solution. If current flows among the cells an electric arch is formed and the cells are shocked and so killed. The buffer for the T4 ligase enzyme is formed, among other chemicals by salts, needed by the enzyme to catalyze the reaction. This created an electric arch during the electroporation that killed the majority of the cells. The few that survived were screened via colony PCR as no other, and possibly easier, screening procedure was viable. This is a direct cause of the fact that A. baumannii, is not a model organism, so it inherits an intrinsically complex genome and structure that complicate the screening techniques. These two critical constraints pushed us to consider other options to further try and clone the dCas sequence in this new plasmid backbone.

Cycle three

Design

Given the results of the previous experiments and attempts, we decided to take a straightforward approach by directly changing the E. coli Origin of replication and substituting it with a medium copy ORI. Since the plasmid from which we extract the dCas expression cassette carries a p15a ORI and we know it allows for dCas expression in E. coli, we decided that this sequence could be a possible solution. We decided then to exchange the plasmid pMB1 ORI with p15a. To operate this modification we opted for a Gibson assembly, for which we designed appropriate PCR primers pairs to amplify the plasmid backbone and the new ORI. After changing the ORI we would attempt to clone the dCas9 between the prefix and suffix sequences and obtain colonies.

Build

We performed two PCR mutagenesis with flanking primes and then assembled the two purified constructs, through Gibson Assembly kit (GeneArt, ThermoFischer Scientific). The product of the reaction was transformed in chemically competent E. coli cells and plated on selective LB agar plates. After overnight incubation the grown colonies were picked and inoculated in selective liquid LB nutrient broth to assess for the correct assembly. By taking advantage of the prefix and suffix sequences we attempted once again to clone the dCas construct (part BBa_K4727008).

Test

We performed two different tests to assess the correct insertion of the new ORI, the first one, being the sequencing of the mutated region, the second being a plate reader experiment to verify the alteration in RFP expression levels in E. coli due to reduced number of plasmid copies.The sequencing gave us positive results. We also attempted the clonage of the dCas expression cassette. After the cloning few colonies could be seen.

Learn

The new plasmid backbone, with the p15a ORI for E. coli has been deposited in the registry as part BBa_K4727009. As few colonies grew on the transformation plate, we learned that the copy number of the initial ORI was not compatible with the toxicity of the dCas9 protein in E. coli.

A matter of promoters

Cycle one

Design

While designing an expression cassette for our newly introduced plasmid backbone, BBa_K4727000, we wanted to achieve high expression levels in multiple hosts, namely E. coli, K. pneumoniae and A. baumannii, as this construct is intended to function in all of them. For these reasons or first design presented a double promoter setting, constituted by the strong promoter BBa_J23119, known to work both in A. baumannii and K. pneumoniae, and the strong promoter pTet (BBa_B0040).

Build

This design idea has been implemented to be tested with a reporter gene, RFP, prior to the assembly of the intended expression cassette for dCas9. This was supposed to be a preliminary test to further investigate the possibility of silencing genes in the target species. As specified in the notebook section the double promoter system was cloned in BBa_K4727000 upstream the RPF gene (BBa_E1010). The expression cassette was completed with a double terminator BBa_B0010 and BBa_B0012.

Test

After cloning the so build construct, both in E. coli and A. baumannii, no red colonies could be seen in the plated bacteria. Further assessment of the transformed bacteria was made with a plate reader experiment to further understand if low level expression was present, but no signal could be revealed.

Learn

This expression cassette is not working as intended so we can conclude that, since the RFP, and the double terminators are already known to work within E. coli in the same setting here tested, there could be a problem regarding the double promoter arrangement.

Cycle two

Design

Given the results of the previous experiment, we decided to separate the promoters and design two different expression cassettes with the single promoter in each one.

Build

Following a standard cloning protocol we cloned in BBa_K4727000 the expression of the RFP under the regulation of BBa_J23119 and, in parallel, of pTET (BBa_B0040).

Test

After cloning the construct bearing BBa_J23119 in E. coli, A. baumannii and K. pneumoniae, red colonies could be seen in the plated bacteria. Further assessment of the transformed bacteria was made with a plate reader experiment to assess and compare expression levels with standard references. Similarly the pSGAb plasmid bearing the pTet promoter was transformed into E. coli and A. baumannii to assess RFP expression. To our great surprise, the pTet promoter showed significant expression levels also in A. baumannii.

Learn

Whereas it is widely known that these two promoters allow expression in E. coli, we tested them in A. baumannii and K. pneumoniae. Whereas we knew, from the current literature that part BBa_J23119, could work in A. baumannii and K. pneumoniaee (see our contribution), we caught with great surprise the news of pTet expressing proteins in A. baumannii.

References

1. S. A. Benner and A. M. Sismour, ‘Synthetic biology’, Nat. Rev. Genet., vol. 6, no. 7, Art. no. 7, Jul. 2005, doi: 10.1038/nrg1637.