Parts
@Sakura Grant @Michelle Anne Hughes
General Overview:
Parts Main Page: http://parts.igem.org/Main_Page
Criteria:
https://2020.igem.org/Judging/Medals
Bronze:
Make a useful contribution for future iGEM teams.
Required URL: 2020.igem.org/Team:YourTeamName/Contribution
Some ways to achieve this include: • Add new documentation to an existing Part on that Part's Registry page: • This could be new information learned from literature • This could be new data collected from laboratory experiments • Build upon an existing software or hardware tool • Document troubleshooting that would be helpful to future teams • Create a 3D printed piece of hardware and document how to make it We invite you to also think outside of these areas for your contribution.
Silver:
Engineering Success
Demonstrate engineering success in at least one aspect of your project. This achievement should be distinct from your Contribution for Bronze.
Required URL: 2020.igem.org/Team:YourTeamName/Engineering
Engineering success can be achieved by making an effort to follow the engineering design cycle:
Research → Imagine → Design → Build → Test → Learn → Improve → Research...
- We invite you to think about ways to tackle and solve one or more of your project's problems and use synthetic biology tools to generate expected results.
- If you are unable to get into a lab, how would you design your experiments, evaluate the outcome, deal with unexpected results, and plan further steps?
Notes:
- For teams who can get into lab, you can design and build a new Part and show that it works as expected (documentation must be on the Part's Pages on the Registry)
Gold:
Improvement of an Existing Part
Make a new Part that improves the function of an existing Part. This improvement must be distinct from your work for Bronze and Silver medals. Team to enter the Existing Part Number and the New Part Number in their Judging Form.
Some things to consider when designing and showing your improvement: • Your experiments should be done with both the improved part and the original part as a control • The sequences of the new and existing parts must be different • Adapting the part to a different assembly standard does not count as a functional improvement • See the Measurement Hub for details on how to measure your parts • You must document the improvement on the Registry on both the existing and new part pages. See the Registry Document Parts page for instructions Notes: • This criteria was kept in as an option for teams who could get into the lab.
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).
Examining efficiency of ribosomal RNA operon B (rrnB) T1 terminator from E. coli. (Schmidt et. al., 1987):
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.
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:
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.
TDMH (Trehalose Dimycolate Hydrolase)
https://2013.igem.org/Team:Paris_Bettencourt
This serves as an alternative/additive addition to the previous parts addition to fulfill the bronze category.
2013 original part's posting: http://parts.igem.org/wiki/index.php?title=Part:BBa_K1137008
- direct quote "Trehalose Dimycolate Hydrolase (TDMH) is a cutinase-like serine esterase that triggers rapid lysis of the mycobacterial cell wall. This enzyme was first isolated form M. smegmatis mutant strain and it could hydrolyze purified TDM from various mycobacterial species. It was show that exposure to TDMH triggers an immediate release of free mycolic acids from noncovalently associated mycolyl-containing glycolipids, ultimately leading to rapid and extensive lysis of pathogenic species, such as M. tuberculosis, M. bovis, and M. marinum, as well as to a lesser extent of M. smegmatis and M. avium (Yang et al. 2012)."
Contribution
TMDH hydrolyzes the TDM found in the mycomembrane (myobacterial outer membrane of Mycobacterium tuberculosis and related pathogens). By doing so, it allows greater nutrient absorption but puts the bacterium susceptible to the stress of the host. This means it plays a critical role in regulating the pathogen's intracellular growth. Tight regulation of the substance occurs else cell lysis occurs.
- "trehalose dimycolate (TDM), a major glycolipid of the mycomembrane, is broken down by the mycobacteria-specific enzyme TDM hydrolase (Tdmh) in response to nutrient deprivation, a process which appears to modulate the mycomembrane to increase nutrient acquisition, but at the expense of stress tolerance"
- "Tdmh is induced during nutrient deprivation and degrades TDM to increase permeability of the cell envelope toward nutrients, while concomitantly lowering the bacterium’s defenses and sensitizing it to stress"
- TDMH degrades TDM to release free mycolic acid
- "when recombinant Tdmh was exogenously added to cells, TDM was degraded to the point of cell lysis, implying that mycobacteria must tightly regulate the concentration of active Tdmh to avoid autolysis while also allowing for beneficial mycomembrane remodeling to occur"
- Investigation of Tdmh's role in Mycobacterium tuberculosis (Mtb)
- Too much tdmh leads to cell rupture: "An uncontrolled exogenous exposure of mycobacteria to a serine hydrolase of TDM from M. smegmatis, called TdmhMs, leads to a rapid depletion of trehalose mycolates and subsequent rupturing of the envelope"
- "TdmhMtb provides a growth advantage to intracellular Mtb in an immunocompromised host."
References
| Author, Year | Link | Column |
|---|---|---|
| Untitled | ||
| Schmidt, 1987 | https://doi.org/10.1016/0022-2836(87)90486-4 | 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. |
| Chamberlin, 1997 | https://doi.org/10.1073/pnas.94.25.13548 | 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. https://doi.org/10.1073/pnas.94.25.1354 |
| Nojima, 2005 | https://doi.org/10.2116/analsci.21.1479 | 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 et. al, 1991 | https://febs.onlinelibrary.wiley.com/doi/pdfdirect/10.1111/j.1432-1033.1991.tb16326.x | 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. |
| Holmes et. al., 2019 | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6396954/ | A FRET-Based Fluorogenic Trehalose Dimycolate Analogue for Probing Mycomembrane-Remodeling Enzymes of Mycobacteria |
| Yang et. al., 2014 | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3974621/ | A hydrolase of trehalose dimycolate induces nutrient influx and stress sensitivity to balance intracellular growth of Mycobacterium tuberculosis |