Team:Uppsala University/Project

Team Uppsala University 2012 is dedicated to combating the rising antibiotic resistance in bacteria by means of synthetic biology. Old and well-known antibiotics are quickly becoming ineffective as resistance genes are spreading. Scientist around the world struggle with varying success to develop new antibacterial substances. But do we really have to abandon classic antibiotics? Team Uppsala University begs to differ, we believe new methods will allow us to combat the resistance itself, and make the bacteria once again sensitive to old drugs.

Working with real-world resistance genes isolated from ESBL outbreaks at Swedish hospitals, we are developing anti-resistance systems active at three different levels: DNA level, transcriptional level and translational level. Our systems will be delivered to the target bacteria using an engineered phage and/or a conjugative plasmid.

At DNA level, we will develop a method for permanent removal of plasmids from bacteria. Using TAL Effector Nucleases, we will be able to target and cut individual resistance genes.

At transcriptional level, we will use synthetic super-repressors to repress transcription of resistance genes and native defense mechanisms in bacteria.

At translational level, we will construct a modular large-scale screening system for sRNA:s and use it to find strongly silencing RNA sequences against three common resistance genes.

With this team on the project, there is no question about it:''' Resistance is futile!'''

Challenge
The primary goal was to construct synthetic small RNAs to silence antibiotic resistance genes. This could potentially be a way to make resistant bacteria sensitive against existing antibiotics, but the problem is to design or find the ideal sRNA sequences.

Background
Spot42 is one of many non-coding RNAs in Escherichia coli. It is a small regulatory RNA that acts as one of the factors involved in the central and secondary metabolism where it regulates the switch between fermentation and respiration [2]. Spot42 contains two distinctive regions, where one is the antisense region, the part of the sRNA that interacts with other RNAs, and one is the region that recruits an RNA binding protein called Hfq.

Our goal was to engineer the native sRNA spot42 to instead target the kanamycin resistance gene AAC(6’), isolated from an ESBL plasmid from an outbreak of multiresistent bacteria in a hospital in Sweden. In theory, the sRNA with its modified antisense region would Watson-crick base pair with the complementary mRNA sequence at the 5’UTR of the antibiotic resistance gene AAC(6´), blocking ribosomal binding. This would supposedly lead to an inhibition of the translation and henceforth a silencing of the antibiotic resistance gene AAC (6´).

Earlier studies of how to design an artificial sRNA showed that there were unknown factors determining whether an sRNA would be effective in blocking translation or not. To find the optimal sRNA, a large randomised library of sRNAs was made to find sequences efficient enough to down regulate the antibiotic resistance with a combinatorial approach[1].

Constructing a randomized library of small RNAs The native Spot42 gene spf from E coli was cloned in a BioBrick plasmid, and placed in front of a synthetic constitutive promoter (J23101). Using this plasmid as template, primers binding to the Hfq binding region and the promoter with overhangs containing a randomized nucleotide sequence of 30 bp was designed (15 randomized nucleotides per primer). By running an inverse PCR on the plasmid with these primers and religating the mutagenized plasmids, a randomized library was created with a maximal theoretical size of 4^30 unique sRNA, only limited by the volume of the PCR reaction.

In order to screen for small RNAs with an antisense region hybridizing in an silencing manner to the 5’ UTR of the antibiotic resistance gene AAC (6´), the randomized sRNAs were transformed into an E coli MG1655 strain carrying a reporter system containing the native 5´UTR of AAC(6’) followed by an additional 15 codons of the coding sequence of AAC(6’) translationally fused via a linker (J18922) to the yellow fluorescent protein SYFP2 (K864100).

Since the reporter system and the sRNA library were on two different plasmids, two suitable plasmid backbones were chosen from different compatibility groups. To make the reporter system as similar to the expression levels of the natural resistance genes, a low copy origin such as pSC101 (BBa_K864001) and the sRNA library required a medium copy backbone such as p15A. Unfortunately, the low copy backbones of the pSB4X5 serie in the registry did not display the predicted behavior of a low copy plasmid, henceforth the decision to construct new plasmid backbones that could meet our requirements Read more

A randomized antisense region of thirty bases resulted in an immense library of sRNAs, where the limiting factor was the transformation efficiency of or competent cells. To be able to find promising silencing sRNAs in this vast library, a Fluorescence Activated Cell Sorter (FACSAria II from BD) was used to sort out 20 000 cells that showed a downregulation of SYFP2 from a total of 10^7 cells. By using the cell sorter function on the FACS machine the amount of false postives were dramatically reduced. This is because the FACS it makes it possible to differentiate between cells with a very low fluorescence and cells that have no fluorescence at all, non-fluorescent cells were expected to might have picked up a loss of function mutations in the SYFP gene.

The cells sorted based on lowered fluorescence were plated on selective agar plates and studied under UV light in order to screen for colonies containing a small RNA that had downregulated the SYFP2 gene. This was problematic due to radiative DNA-damage that was inflicted on the bacteria, and a substantial difference in cell growth was observed. This problem was resolved by switching to a Visi-Blue transilluminator, avoiding damage to the cells and also simplified future screening.

Clones that showed lowered expression of SYFP2 were measured for fluorescence using flow cytometry. The fluorescence levels could be distinguished with a accuracy of just a few percent.

The plasmids that contained sRNA down regulating SYFP2 were purified and transformed into DH5alpha, and then purified again to ensure pure plasmid clones free from reporter plasmids.

Finally, the isolated sRNA plasmids were transformed into a new reporter strain to validate the down regulation with flow cytometry

The reporter system together with the native spot42 has been sent as parts to the registry. This is to give future iGEM teams the possibility to repress any gene by replacing the RFP region with the 5´UTR of the gene of interest.