Coding

Part:BBa_K2560123

Designed by: Tobias Hensel   Group: iGEM18_Marburg   (2018-09-22)


Phytobrick version of pTet

This is the Phytobrick version of the promoter pTet and was build as a part of the Marburg Collection. Instructions of how to use the Marburg Collection are provided at the bottom of the page.

Overview

Promoters are genetic modules were the RNA polymerase is recruited to start RNA transcription. They are divided in two groups: constitutive promoters which transcribe RNA permanently and inducible promoters which start the transcription as a response to a stimulus. Inducible promoters can be regulated by transcription activation or repression. To start the RNA transcription the RNA polymerase complex is not sufficient. Therefore sigma70 factors are required. The sigma70 factor binds to the Pribnow box – two motifs -10 and -35bp upstream the CDS – recruiting the RNA transcrition complex enabling the transcription (Huerta and Collado, 2003), ( Paget and Helmann, 2003). Based on this knowledge a collection of synthetic constitutive promoters have been developed by Chris Anderson and made available on the iGEM repository.

Characterization

Anderson Promoters


T--Marburg--LUXPicture new.png


BerkiGEM2006-Promoters.jpg


 Variant Lux (au)
 
 K2560131 (Dummy)    0.025
 K2560019 (J23103)   0.032
 K2560026 (J23113)   0,038
 K2560023 (J23109)   0,052
 K2560009 (J23104)   0,058
 K2560029 (J23117)   0,090
 K2560025 (J23111)   0,098
 K2560028 (J23116)   0,134
 K2560021 (J23107)   0,136
 K2560027 (J23114)   0,163
 K2560024 (J23110)   0,169
 K2560018 (J23102)   0,245
 K2560030 (J23118)   0,348
 K2560020 (J23105)   0,387
 K2560015 (J23115)   0,398
 K2560014 (J23106)   0,502
 K2560017 (J23101)   0,510
 K2560022 (J23108)   0,768
 K2560007 (J23100)   1


We started by measuring the promoter strength of the Anderson Promoter library in V. natriegens. Firstly, we assembled 19 test plasmids with golden-gate-assembly and measured their expression strength, following our selfmade workflows. The results are shown in Figure 1. We observed an even distribution of the tested promoters throughout the dynamic range. The strongest promoter K2560007 (J23100) yielded 40 fold stronger signal than the promoter dummy and was used as a reference to calculate relative promoter strengths.
The test constructs were built with dummy connectors which did not possess insulator elements. We assume that this resulted in additional expression caused by transcription throughout the rest of the plasmid, e.g. ori and antibiotic resistance. This is thought to add the same extent of signal to all measured promoters thus reducing the overall dynamic range. To further evaluate this assumption, we could repeat this experiment with one of our insulators instead of the dummy connector.

Figure 1: Relative promoter strenghts of promoters from the Anderson Collection in Vibrio natriegens.
The promoter test construct is shown in Figure 2. Contruct were messured in quadruplicates and in three indipendent experiments.
Figure 2: Contruct for messuring promoter strenghts.
The label "Tested Promoters" is a place holder for the respective promoter. The plasmid was assembled from eight basic parts by Golden Gate Assembly.

Inducible Promoters

In addition to constitutive promoters, the Marburg Collection contains two inducible promoters, pTet and pTrc. For all experiments with inducible promoters, we added the respective inducer concentration to the preculture as well as to the main culture to ensure constant expression.The first experiments were performed with the pTet promoter that can be induced by the tetracycline derivative anhydrotetracycline (ATc). ATc is much less cytotoxic but still capable of binding and altering the structure of the repressor TetR, leading to release of the promoter and enabling transcription. To measure the dose response behavior of the pTet, we made a dilution series of ATc. Following the recommendation of our advisors (Stefano Vecchione), we started with the concentration commonly used in E. coli, started with the concentration (100 ng/mL). The starting concentration was diluted twofold in 20 subsequent steps. Our results are shown in figure 3. The absence of bars for the four highest concentrations means that the cultures did not reach an OD of 0.2 in the seven hours of the measurement. Remarkably, we observed reasonable growth of those same cultures in the preculture already induced with the identical amount of ATc. Knowing that luminescence is produced at the end of an enzymatic cascade, starting with intermediates of the phospholipid metabolism ( Meighen 1991), we reckon that very strong induction could decrease the fitness of cells and that after dilution in room temperature medium, strained cells are not able to recover from the stationary phase. However, we only observed this phenomenon in experiments with pTet, although we obtained higher signals for the strongest constitutive promoters as well as for the highly induced pTrc. We checked for toxicity of ATc but could not see a measurable effect (figure 4). Another possibility is that TetR interacts with components inside the cell and that high ATc increases these interactions. Blast searches of TetR against the genome of V. natriegens identified one protein that shares some homology with the N-terminal part of TetR which could result in cross talk between the host and the inducible promoter.



Figure 3: Dose response of pTet with ATc.
J23100 was used as positive control and for normalization. Error bars represent the standard deviation of the measurements of three independent experiments


All measured data were normalized to the strongest constitutive promoter K2560007 (J23100). Saturation occurred at a dilution of 2^6 (~ 1.6 ng/mL) and an exponential reduction of luminescence signal can be observed for higher dilutions. In the absence of ATc, the signal is twelve fold lower compared to saturation.
pTet allows relatively tight control of gene expression and is therefore well suited for driving the expression of potentially toxic proteins. On the other hand, we were not able to induce strong expression that can compete with strong constitutive promoters or the fully induced pTrc.
pTrc is the second tested inducible promoter. It contains lac operator sites and is therefore regulated by the repressor LacI which is constitutively expressed from a downstream gene. pTrc can be induced Isoopropyl-β-D-thiogalactopyranosid (IPTG), a chemical derivative of lactose ( Camsund et al.2014). Similar to our experiments with pTet, we made a dilution series starting with the commonly used IPTG concentration for E. coli 0.5 mM. We observed a five fold induction and a saturation that occurred at a dilution of 2^5 (~15 µM). The strongest expression is similar to the expression gained from the strongest constitutive promoter J23100 while the expression in the absence of inducer equals medium strong promoters. As a consequence, we do not recommend using pTrc in constructs where a tight control of gene expression is desired. However, pTrc is well suited when strong expression is required.

Figure 4: Dose response of pTrc with IPTG.
J23100 was used as positive control and for normalization. Error bars represent the standard deviation of the measurements of three independent experiments

Taking the results of both inducible promoters into account, we made two observation. In both cases, the dynamic range is smaller compared to E. coli and the inducer concentration that facilitates saturation is 32 and 64 fold lower for pTrc and pTet, respectively, than the concentration that is typically used for E. coli. A possible explanation could be found in the fast growth of V. natriegens which might result in a lower concentration of the repressor proteins in the cells, finally leading to a less restricted control of the negatively regulated promoters. However, we do not have experimental support for our idea.

Usage and Biology

Marburg 2018 characterized this part in Vibrio natriegens using the lux operon of Photorhabdus luminescens (BBa_K2560051). The parts sequence was verified by Sanger sequencing.

Sequence and Features

Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]


Marburg Toolbox

We proudly present the Marburg Collection, a novel golden-gate-based toolbox containing various parts that are compatible with the PhytoBrick system and MoClo. Compared to other bacterial toolboxes, the Marburg Collection shines with superior flexibility. We overcame the rigid paradigm of plasmid construction - thinking in fixed backbone and insert categories - by achieving complete de novo assembly of plasmids.

36 connectors facilitate flexible cloning of multigene constructs and even allow for the inversion of individual transcription units. Additionally, our connectors function as insulators to avoid undesired crosstalk.

The Marburg Collection contains 123 parts in total, including:
inducible promoters, reporters, fluorescence and epitope tags, oris, resistance cassettes and genome engineering tools. To increase the value of the Marburg Collection, we additionally provide detailed experimental characterization for V. natriegens and a supportive software. We aspire availability of our toolbox for future iGEM teams to empower accelerated progression in their ambitious projects.


Figure 3: Hierarchical cloning is facilitated by subsequent Golden Gate reactions.
Basic building blocks like promoters or terminators are stored in level 0 plasmids. Parts from each category of our collection can be chosen to built level 1 plasmids harboring a single transcription unit. Up to five transcription units can be assembled into a level 2 plasmid.
Figure 4: Additional bases and fusion sites ensure correct spacing and allow tags.
Between some parts, additional base pairs were integrated to ensure correct spacing and to maintain the triplet code. We expanded our toolbox by providing N- and C- terminal tags by creating novel fusions and splitting the CDS and terminator part, respectively.


Parts of the Marburg Toolbox




Tags and Entry Vectors




  • K2560001 (Entry Vector with RFP dropout)
  • K2560002 (Entry Vector with GFP dropout)
  • K2560005 (Resistance Entry Vector with RFP Dropout)
  • K2560006 (Resistance Entry Vector with GFP Dropout)
  • K2560305 (gRNA Entry Vector with GFP Dropout)