Part Improvement - Extending the Registry
A core conviction of iGEM is that of modularity. With an extensive bank of parts, others can find and use existing parts to aid their project, extend the knowledge of existing parts, and add new parts.
Since the onset of the Covid-19 pandemic, many iGEM participants have been unable to go to lab spaces to run experiments. It was during this time that our team decided to further enhance an existing part on the registry in the hopes that future participants would gain value from them.
As at the time our team was unable to access lab spaces, we redirected our energy toward making meaningful contributions from information learned from literature. We uncovered and contributed to two interesting parts: the Trehalose Dimycolate Hydrolase enzyme and the T1 terminator from E. Coli rrnB.
Trehalose Dimycolate Hydrolase (TDMH)
We came across and were excited by a coding part for a cutinase-like serine esterase. In 2013, Paris Bettencourt contributed a part for Trehalose Dimycolate Hydrolase (TDMH) which functions to trigger rapid lysis of the mycobacterial cell wall of mycobacteria. It accomplishes this by hydrolyzing TDM and releasing free mycolic acids from mycolyl-containing glycolipids, disrupting the mycomembrane’s integrity Yang et. al., 2014. The team investigated the efficiency of TDMH producing E. Coli on lysing and killing mycobacteria.
Simplified model for mycomembrane remodeling (B) Biosynthesis and breakdown of TDM. Holmes et. al., 2019
While diverging from the work our team was doing, we found the part intriguing for its ability to induce mycobacterial cell wall lysis. We investigated literature further and found promising new papers exploring endogenous TDMH regulation in mycobacteria. While the previous iGEM team showed high TDMH concentration introduced to the mycobacteria causes lysis, it has been shown mycobacteria produce TDMH to regulate nutrient absorption. TDM is a major glycolipid of the mycomembrane. TDMH breaks TDM down in nutrient limiting environments, modulating the mycomembrane to enhance nutrient acquisition. However, by this same process, the mycobacteria become more susceptible to stress from the host’s immune response Holmes et. al., 2019 . What follows is the contribution we made to the part registry:
It was shown that the concentration of TDMH is tightly regulated in the mycobacteria to balance nutrient absorption and susceptibility to stress from the host’s immune response Holmes et. al., 2019 . While exposure to high levels of exogenously introduced TDMH has been shown to trigger cell lysis in mycobacterium, these pathogens endogenously produce TDMH in response to nutrient depravation. The breakdown of TDM in the mycomembrane increases the membrane’s permeability to nutrient intake, but concurrently sensitizes it to stress of the host. It was shown TDMH confers a growth advantage to intracellular Mtb in MyD88−/− mice hosts. A study showed Rv3451 is the primary TDMH of mycobacterium tuberculosis (TdmhMtb). TdmhMtb responds to the host’s immunity by regulating Mtb growth, providing a growth advantage to Mtb in an immunocompromised host Yang et. al., 2014.
T1 terminator from E. coli rrnB (Part BBa_B0010)
Transcription of DNA is made up of the following steps: RNA polymerase binding to promoter and activation, initiation of RNA transcript, elongation of RNA transcript, and termination of transcription. Termination is the last step of transcription where the RNA polymerase releases the RNA transcript. Releasing the RNA transcript is not reversible and further transcription requires reinitiation at a promoter region to form a new RNA transcript (Uptain et. al, 1997). In prokaryotes like E. coli, the termination sites serve as targets for gene expression regulation as they can not only occur at the end of genes, but also near promoter regions or between genes in the operon (Nojima et. al., 2005). The T1 terminator region on the rrnB gene of E. coli is one of two terminator regions, the other being T2. The two termination regions have "factor-independent terminator-like sequences" with "two additional inverted repeats (IR1 and IR2) and a pair of direct repeats" (Orosz et. al., 1991). The T1 and T2 terminating regions are often used as an efficient terminator of transcription in many cloning vectors (Orosz et. al., 1991).
The contribution for this part is in examining efficiency of ribosomal RNA operon B (rrnB) T1 terminator from E. coli:
Efficiency is enhanced by the E. coli nusA protein, which gives effectiveness of inhibition in vitro comparable to those in vivo. These transcripts that are terminated when nusA protein is present are released from the RNA polymerase complex, suggesting that there is a complete termination reaction. The protein's termination factor activity is not dependent on the presence of the rho protein. The nusA protein serves as an antitermination factor, RNA polymerase subunit, and true termination factor at some terminator sites. In general, termination at T1 in vitro is quite efficient with an 80% effectiveness rate without any additional factors. In vivo and in E. coli extracts, the T1 terminator has shown to be nearly 100% efficient. In an isolated and purified system with only nusA protein present, termination at this high level of efficiency is also achieved, suggesting that in vivo, the entity responsible for the highly efficient termination is due to the nusA protein (Schmidt et. al., 1987).
The intrinsic terminator is composed of the NusA protein, which interacts with the new RNA exiting out of the channel to stimulate termination. (Santangelo et. al., 2011)
Literature used - TDMH:
Holmes, N. J., Kavunja, H. W., Yang, Y., Vannest, B. D., Ramsey, C. N., Gepford, D. M., Banahene, N., Poston, A. W., Piligian, B. F., Ronning, D. R., Ojha, A. K., & Swarts, B. M. (2019). A FRET-Based Fluorogenic Trehalose Dimycolate Analogue for Probing Mycomembrane-Remodeling Enzymes of Mycobacteria. ACS omega, 4(2), 4348–4359. https://doi.org/10.1021/acsomega.9b00130
Yang, Y., Kulka, K., Montelaro, R. C., Reinhart, T. A., Sissons, J., Aderem, A., & Ojha, A. K. (2014). A hydrolase of trehalose dimycolate induces nutrient influx and stress sensitivity to balance intracellular growth of Mycobacterium tuberculosis. Cell host & microbe, 15(2), 153–163. https://doi.org/10.1016/j.chom.2014.01.008
Literature used - T1 terminator:
Nojima, T., A. C. Lin, T. Fujii and I. Endo (2005). "Determination of the Termination Efficiency of the Transcription Terminator Using Different Fluorescent Profiles in Green Fluorescent Protein Mutants." Analytical Sciences 21(12): 1479-1481.Nojima, T.; Lin, A. C.; Fujii, T.; Endo, I., Determination of the Termination Efficiency of the Transcription Terminator Using Different Fluorescent Profiles in Green Fluorescent Protein Mutants. Analytical Sciences 2005, 21 (12), 1479-1481.
Orosz, A., I. BOROS and P. VENETIANER (1991). "Analysis of the complex transcription termination region of the Escherichia coli rrn B gene." European journal of biochemistry 201(3): 653-659.
Schmidt, M. C. and M. J. Chamberlin (1987). "nusA Protein of Escherichia coli is an efficient transcription termination factor for certain terminator sites." Journal of Molecular Biology 195(4): 809-818.
Uptain, S. M., & Chamberlin, M. J. (1997). Escherichia coli RNA polymerase terminates transcription efficiently at rho-independent terminators on single-stranded DNA templates. Proceedings of the National Academy of Sciences of the United States of America, 94(25), 13548–13553.
Santangelo, T. J. and I. Artsimovitch (2011). "Termination and antitermination: RNA polymerase runs a stop sign." Nature reviews. Microbiology 9(5): 319-329.