Team:SVCE Chennai/Project
Project: Minicins
Project Design
Our project aims to neuter multidrug resistant bacterial infections with the help of bacteriocins in a minicell chassis.
Firstly, the idea was to use two different plasmids – one for minicell division and the other for bacteriocin production. Then, we realized that plasmids are carried to the minicells. This requires the use of two different plasmids with two different promoters. Instead of going for two different plasmids, we modulated our design to a plasmid with two different expression cassettes.
The two cassettes are under the regulation of different promoters for the expression of the different phenotypes. Minicell phenotype, driven by the overexpression of the ftsZ gene, is under the regulation of the IPTG-controlled lac promoter. Bacteriocin production is driven by the arabinose operon.
In order to confirm the expression of ftsZ, GFP is included under the lac promoter control by the introduction of an RBS after the ftsZ stop codon. Similarly, bacteriocin production is tagged to RFP.
pSB1K3 was preferred to pUC vectors since minicell purification involves the addition of penicillin and that requires penicillin – sensitivestrains. In order to drive very high expression of ftsZ, we decided to use the T7 promoter. It requires T7 RNA polymerase, which also needs to be cloned into the plasmid. ftsZ can be indirectly regulated by controlling the expression of T7 RNA polymerase.
A major drawback that can be seen in this design is that there are over 10 inserts and the total insert size amounts to more than 8 kb.
The number of inserts and their size can be greatly reduced by judicious selection of the vector and the host strain. In order reduce the inserts corresponding to the ftsZ regulation which involves T7 RNA polymerase and promoter, BL21 was chosen as the host strain and pET 24a was identified as the vector. BL21 strain has T7 RNA polymerase inserted into its genomic DNA. pET 24a vector has a multiple cloning region preceded by an operator region consisting of the lac operator and the T7 promoter.
Hence, a second construct was designed utilizing the pET 24a vector. The complete insert was to be inserted between the NdeI and XhoI restriction sites. After assembly of the parts by standard biobrick assembly, the complete insert will be PCR amplified with NdeI and XhoI sites flanking the entire insert.
The sequences for both the cassettes were ordered as gBlocks® from IDT. The complete cassette was divided into two gBlocks® sized 1664 bp and 1057 bp respectively. A SacI site was introduced in the araC region of the pBAD promoter. The nucleotide in position 430 was mutated from C to G for creating a unique SacI site. The mutation created no changes in the 3’- 5’ araC reading frame.
The gBlocks® received were PCR amplified. No amplification was detected for gBlock® 1. We deduced that the gBlock® had denatured into several small fragments by agarose gel electrophoresis. Hence, we set out for standard biobrick assembly to assemble ftsZ, T0 terminator and pBAD/araC promoter. The block 1 was constructed by assembling every component. PCR amplification was then done to introduce the NdeI and SacI restriction sites. The gblock® 2 containing the two test bacteriocin sequences were obtained from IDT.
The fragments were cloned one by one in the pET 24a vector.
Protocol
- Spin the cell culture in a centrifuge to pellet the cells, empty the supernatant (media) into a waste collection container.
- Resuspend pelleted bacterial cells in 250 µl Buffer P1 (kept at 4°C) and transfer to a microcentrifuge tube.
- Add 250μl Buffer P2 and gently invert the tube 4–6 times to mix. Do not vortex, as this will result in shearing of genomic DNA.
- Add 350μl Buffer N3 and invert the tube immediately and gently 4–6 times.
- Centrifuge for 10 min at 13,000 rpm (~17,900 x g) in a table-top microcentrifuge. A white pellet will form.
- Apply the supernatants from step 4 to the QIAprep spin column by decanting or pipetting.
- Centrifuge for 30–60 s. Discard the flow-through.
- Wash the QIAprep spin column by adding 0.5ml Buffer PB and centrifuge for 30-60s. Discard the flow-through.
- Wash QIAprep spin column by adding 0.75 ml Buffer PE and centrifuging for 30–60s.
Spinning for 60 seconds produces good results. - Discard the flow-through, and centrifuge for an additional 1 min to remove residual wash buffer.
- Place the QIAprep column in a clean 1.5 ml microcentrifuge tube. To elute DNA, add 50μl Buffer EB (10 mM Tris·Cl, pH 8.5) or water to the center of each QIAprep spin column, let stand for 1 min, and centrifuge for 1 min.
1. Excise the dna fragment from the gel with a clean, sharp scalpel. Weigh the gel slice and transfer to a 1.5 ml microfuge tube.
2. Add 400µl of binding buffer 2 for each 100mg of gel weight. Incubate at 50˚C to 60˚ for 10mins and shake occasionally until the agarose is completely dissolved. For high concentration gels 700µl of binding buffer 2 per mg of agarose gel are added.
3. Add the above mixture to the EZ-10 column and let stand for two minutes. Centrifuge at 10,000 rpm for two minutes and discard the flow-through in the tube.
4. Add 750 µl of wash solution and centrifuge at 10,000rpm for 1 minute. Discard the solution in the tube.
5. Repeat step 4. Centrifuge at 10,000rpm for an additional minute to remove any residual wash buffer.
6. Place the column in a clean 1.5 ml microfuge tube. Add 30-50 µl of elution buffer to the centre of the column and incubate at room temperature for 2minutes. Centrifuge at 10,000rpm for 2minutes to elute the dna.
7. Store purified dna at -20˚C.