By Hansub Kim
Research on the genetic response of bacteria towards bacteriophages and other viruses can lead to new insights in virology and synthetic biology. This experiment was designed in order to quantify the effectiveness of inhibition of bacteriophage Lambda against E. coli through inhibition of the replication site. Phage Lambda has been studied extensively in the past, making it a good platform for generalization.
First. DH5a E. Coli bacteria was first grown in LB agar plates – 3 control plates and 3 plates with kanamycin resistance for plasmid (pHN1257, received from Dr. Liam Good, Royal Veterinary College, London), and bacteria was transformed. Kan-resistant bacteria was grown in an incubator for 16-20 hours, before performing a miniprep on the kanamycin-resistant E. coli using the Qiagen Miniprep Protocol. Results were observed after 16 hours, and genomic content was recorded with a NanoDrop at Caltech. Upon sufficient genomic concentration, antisense RNA was assembled using custom-designed gene blocks from IDT, and the pHN1257 plasmid was digested into the IDT gblock. Next, the plasmid was ligated with the TaKaRa ligation protocol. and the antisense RNA-fitted plasmid was transformed into the E. coli. Finally, one sample of bacteria with pHN1257 with phage-lambda, and one sample of bacteria without the plasmid were infected respectively, and it is expected that phage lambda replication will be inhibited in the E. coli with the asRNA-fitted pHN1257 plasmid.
If I design an asRNA against genes critical for phage lambda replication by targeting their P protein (replication) site, we will be able to prevent phage growth within E. coli.
The goal of this project was to test the effectiveness of viral replication in bacteria, using antisense RNA as a potential novel mechanism. asRNA, when introduced into a genome, can obstruct translation of an mRNA by base pairing to it. If asRNA is successful in inhibiting the replication of phage Lambda in E. Coli via binding to the mRNA strand coding for the P protein site (which controls replication), it will likely be able to execute similar genetic modifications not just in phage lambda, but in other bacteriophages as well. This project is part of a larger ultimate goal to use asRNA, or a similar mechanism to achieve viral replication inhibition in human gut bacteria for a range of viruses. I eventually hope to create a drug, edible probiotic, or supplement that executes this.
MATERIALS AND METHODS
- Liquid LB 14. Centrifuge
- DI (Deionized Water) 15. Kanamycin Sulfate (Antibiotic)
- Tris-EDTA – Buffer 16. Bacto Agar
- DH5alpha, C600, W3104, Top10 E. coli 17. Incubator
- Nitrile gloves 18. Transformation Solution (CaCl2)
- Safety goggles 19. RNAse A, LyseBlue, various Buffers
- 10uL, 1000uL micropipettes 20. Microcentrifuge Tubes
- Parafilm, shrink wrap, tape 21. Spin Columns
- NanoDrop nanophotometer 22. pHN1257 (plasmid)
- asRNA gBlocks 23. Gel electrophoresis system
- Tryptone 24. PCR Thermocycler
- Yeast Extract 25. Agarose
- Sterile Glass Beads 26. 4-watt General Electric UV lamp
1.) Bacterial Transformation
DH5a E. Coli bacteria was grown in LB agar plates – 3 control plates and 3 plates using kanamycin, an antibiotic, as the selectable resistance marker for plasmid (pHN1257, received from Dr. Liam Good, Royal Veterinary College, London), and bacteria was transformed. The transformed bacteria was grown up on 8 LB/kan plates.
2.) Genomic Purification
A miniprep was performed to purify the plasmid DNA and separate it from the rest of the bacteria.
- Bacteria shaken in an incubator at 37 degrees C for 18 hours, then centrifuged in 2 microcentrifuge tubes to separate the pellet and supernatant.
- Added Buffers P1, P2 vortexing in between
- Washed out the DNA in spin columns, using buffer PB
My samples of purified DNA were later taken to CalTech with the assistance of Dr. Cory Tobin, where genomic content was measured with a nanophotometer. I had to conduct the miniprep over 5 times until I ended up with sufficient genomic content due to the accuracy needed for the procedure.
3.) asRNA assembly
asRNA was designed using the mRNA of the P protein site, which codes for lambda phage replication. I had my asRNA sequence synthesized into a “gBlock” by IDT, pre-prepared for ligation and dried.
The plasmid (pHN1257) was spliced open according to 2 restriction sites that flanked the lambda origin of replication where the gblock could be inserted. The gBlock was then ligated between the two “sticky ends” of the restriction sites (Nco1 and Xho1).
- Digested Nco1 and Xho1 (the two restriction enzyme sites) with 10xready enzymes and water (20uL for plasmid; 10uL for gBLock)
- Let incubate for 1 week.
- Grew up fresh competent cells.
- Created a ligation “mastermix” with 5 parts growth enzyme, 4 parts gBlock digest, and 1 part pHN1257 digest.
- Transformed the ligated pHN1257 competent cells into LB/kan plates with tryptone, yeast extract.
- LB/kan plates were inoculated with the competent cells via sterile glass beads.
- Competent cells grown up in liquid culture; another miniprep performed to purify the plasmid DNA (refer to step 2.)
After the competent cells containing the ligated plasmid were grown, gel digest (gel electrophoresis) was run on the plasmid DNA to determine the length of the DNA, and subsequently, whether or not the antisense RNA gBlock had successfully been incorporated into the plasmid. A reading of 407-408 base pairs meant that the gBlock, ~140bp, had successfully been incorporated.
5.) Phage Induction
- W3104, a strain of E. coli with phage Lambda incorporated in it, was purchased from Carolina Bio, and activated with short wave 4-watt UV light for 30 seconds at 12cm away from the uncovered plate of E. coli.
- Colonies were scraped off with an inoculating loop and mixed with a tris-EDTA buffer, then centrifuged to separate the bacteria (not needed) and the activated virus (needed). Transferred 0.8mL of the supernatant with the virus into 4 microcentrifuge tubes.
- Created 6 plates of LB; took the virus supernatant from last time and created 1x, 100x, and 10,000x dilutions with liquid LB. (C600 – phage susceptible colony, Top10 – colony with the plasmid pHN1257.); created 3 plates with each E. coli colony for 6 plates each
- Created a master mixture of 250uL of bacteria and 50uL of virus – 3 tubes for C600 and Top10 for 6 tubes of master mixture.
- Created 0.7% LB agar (needs to be diluted because of the added bacteria/virus concentration).
- After creating the regular agar plates, 3.5mL of the 0.7% LB agar and the 300uL combined mixture of bacteria and virus (heated up to 37C) were combined, rapidly vortexed, and spread across the regular solidified 1% agar plates to homogenize the mixture, then labelled accordingly.
When a bacterial sample is infected with a bacteriophage, clusters of colonies will die out, leaving rings of dead bacteria called “plaques”. After growing up the 0.7% agar and virus plates, they were observed after 24 hours for plaque formation. At 1x dilutions, the C600 strain, which is normally susceptible to phage Lambda, had a 22 plaque count; the Top10 strand without plasmid (control) had 6 plaques, and the Top10 strand with the pHN1257 plasmid had only 1 plaque, indicating that the asRNA had successfully inhibited phage replication.
For the 1x dilutions, the C600 bacteria strain had a 22 plaque count, the control Top10 had 6, and the Top10 with plasmid had 1, meaning that the incorporation of the plasmid had successfully inhibited the replication of phage Lambda in E. coli. For other dilutions, 100x and 10,000x, plaque count for all was 0, except for the 100x dilution of C600 (susceptible strain). The uncertainty of data in high dilutions of the viral mixture signifies that not much bacteria was activated to begin with, most likely due to the lack of optimal UV viral exposure time specified. Another source of error may have been mathematical error when creating the dilutions. However, data with the original, intended amounts of phage Lambda showed that plasmid-incorporated bacteria produced little to no plaques, meaning that viral replication and activation had been inhibited, which supports my hypothesis.
Based on this study, asRNA-incorporated plasmids that obstruct translation of the replication-coding P protein region of the Lambda genome successfully inhibit replication of bacteriophage lambda, but only in sufficient, proportional concentrations and not in dilutions. The success of this experiment supports the use of asRNA as an effective genetic tool for gene knockout.
Fig. A – Histogram of Bacterial Plaque Count.
Fig B – Raw Data of Plaque Count Fig C – Site of Ligation; Restriction Enzymes
Fig D – Map of Plasmid pHN1257
Fig E. Gel Electrophoresis of pHN1257 DNA
- Nakashima, N., Tamura, T., & Good, L. (2006, October 24). Paired termini stabilize antisense RNAs and enhance conditional gene silencing in Escherichia coli. Retrieved June/July, 2016, from https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1635301/pdf/gkl697.pdf
- Goh, S., Hohmeler, A., Stone, T. C., Offord, V., Sarabia, F., Garcia-Ruiz, C., & Good, L. (2015, August). Silencing of Essential Genes within a Highly Coordinated Operon in Escherichia coli. Retrieved June/July, 2016, from https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4510190/pdf/zam5650.pdf
- Ptashne, M. (2004). A genetic switch: Phage lambda revisited. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press.