Table 1 provides an overview of all the parts (basic and composite) that we created this year.
Our four
main composites belong to our manganese project and were designed to allow for a deeper understanding of
the functionality of the individual parts. Furthermore, we integrated two other composites as a side
project (see
Design).
Table 1: Summary of all basic and composite parts introduced to the BioBrick registry by iGEM
Team Tübingen 2020.
| Biobrick | Name | Type | Description | Length |
|---|---|---|---|---|
| BBa_K3510000 | FAST2-tag | Basic | Dynamic fluorescence-activating tag | 375 |
| BBa_K3510003 | Mn-Promoter-Mn-Riboswitch-FAST-Phytochelatine-Terminator | Composite | Bifunctional manganese biosensor | 1101 |
| BBa_K3510005 | Anderson-Promoter-Mn-Riboswitch-FAST-Phytochelatine-Terminator | Composite | Bifunctional manganese biosensor | 921 |
| BBa_K3510002 | Mn-Promoter-Mn-Riboswitch-FAST-Chromoprotein-Terminator | Composite | Manganese biosensor | 1647 |
| BBa_K3510004 | Anderson-Promoter-Mn-Riboswitch-FAST-Chromoprotein-Terminator | Composite | Manganese biosensor | 1495 |
| BBa_K3510006 | Anderson-Promoter-B12-Riboswitch-Tet-Inverter-System-GFP-Terminator | Composite | Vitamin B12 biosensor | 2245 |
| BBa_K3510007 | Anderson-Promoter-B12-Riboswitch-Tet-Inverter-System-mRFP-Terminator | Composite | Vitamin B12 biosensor | 2209 |
Basic Parts
This part encodes the Fluorescence-Activating and Absorption-Shifting Tag (FAST) protein, a 14 kDA
protein that induces fluorescence in a variety of fluorogens upon assembly with these. This
complementation system allows for detection and imaging with high contrast, as individual fluorogens are
non-fluorescent (1).
The first FAST sequence in the parts registry (BBa_K2992000) was provided by Nottingham (2019). However,
their sequence was codon optimized for use in Clostridium spp. Considering many iGEM teams use E.
coli
as a host for cloning, we saw great value in the addition of a FAST sequence suitable for it. For this,
we acquired the FAST2 from The Twinkle Factory, which contains a single mutation to improve its
fluorescence in comparison to FAST1 [2]. We also integrated a silent mutation to eliminate a PstI
restriction site, which is incompatible with the accepted iGEM standards BioBrick RFC[10] and Type IIS
RFC[1000]. Our modified FAST2 was used for tagging of our proteins of interest: A synthetic
phytochelatin and a blue chromoprotein.
We want to nominate this part as the best basic part, as it allows specific fluorescent tagging of any protein of interest. By using a family of different fluorogens offered by The Twinkle Factory that bind to FAST2, the spectral properties of the fluorescent complex can be exchanged easily, making the experimental set up more flexible (1).
We want to nominate this part as the best basic part, as it allows specific fluorescent tagging of any protein of interest. By using a family of different fluorogens offered by The Twinkle Factory that bind to FAST2, the spectral properties of the fluorescent complex can be exchanged easily, making the experimental set up more flexible (1).
While our initial plan was to use a red fluorescent fluorogen (TFCoral), no new iGEM Measurement Kit was
provided this year due to COVID-19 safety regulations. Therefore, we had to rely on the 2018 kit that
did not include Texas Red for calibration of red fluorescence. Because of the practicality of FAST2, we
simply needed to switch to the fluorogen TFLime (a kind gift from The Twinkle Factory) and calibrate for
green fluorescence with Fluorescein sodium salt according to the iGEM 2019 Plate Reader Fluorescence
Calibration protocol.
In addition, the fluorescent complex between FAST2 and a fluorogen does not rely on molecular
oxygen,
thus enabling fluorescent tagging under anaerobic conditions (1). This would be useful in our experiment
described in
Outlook.
In conclusion, we evaluate this tag as an impressive tool for the synthetic biology toolbox that will be
beneficial for future iGEM Teams, especially those working under low-oxygen conditions (with anaerobic
microorganisms) or those who want to conduct fluorescence measurements under different ventilation
conditions while still obtaining comparable results.
Composite Parts
This composite part is a manganese-inducible expression system of phytochelatin (BBa_K1321005) tagged
with FAST2 (BBa_K3510000). Expression is primarily regulated by two different elements: A
manganese-inducible promoter (BBa_K902073) and a manganese-inducible riboswitch (BBa_K902074).
Expression is regulated on a transcriptional level by the promoter, which is activated by manganese. In
contrast, translation of transcripts is regulated by the riboswitch, which initiates translation only
after interaction with manganese, and would inhibit it otherwise. We combined both elements to tighten
regulation. Transcriptional termination occurs through the activity of the reliable double terminator
(BBa_B0015).
In our project, we coupled this manganese sensing device with a phytochelatin to create a bifunctional
biosensor that would not only allow detection of manganese, but also chelate it, essentially removing it
from the environment. With our suggested implementation, this would contribute
to the improvement of
water quality if applied in a filter system. The FAST2 tag allows a quantitative measurement of
fluorescence, which should provide information about the manganese concentration in a test sample.
Attention: This construct does not include a ribosome binding site (RBS) upstream of the FAST2 sequence.
When building on our design, please make sure you add it in order to allow for translation.
The cloning of this composite part was successful and the steps were perfected (see Notebook)
to
increase the efficiency (Fig. 1). Due to the missing RBS in our design, the results from our
experiments
dependent on protein expression (fluorescence, growth, etc.) are omitted here (see Results),
as they do
not represent the potential functionality of our system.
Figure 1: Colony PCR after Gibson Assembly of Mn-Promoter-Mn-Riboswitch-FAST -Phytochelatine-Terminator (GA2). This figure is an example for the high cloning efficiency when using Gibson Assembly. It also contains samples from two other composite parts (GA3 and GA4).
This composite part is a variant of
BBa_K3510003. In this composite, the Mn-inducible promoter is
replaced by the Anderson promoter, a constitutive promoter. By removing transcriptional regulation,
direct regulation of expression occurs exclusively through the manganese-inducible riboswitch, which
regulates the translation of the mRNA. This allows a closer analysis of the regulatory efficiency of
the riboswitch and compares the functionality of the composite with and without transcriptional
regulation.
Attention: This construct does not include a ribosome binding site (RBS) upstream of the FAST2 sequence.
When building on our design, please make sure you add it in order to allow for translation.
The cloning of this composite part was successful and the steps were perfected (see Notebook)
to
increase the efficiency (Fig. 2). Due to the missing RBS in our design, the results from our
experiments
dependent on protein expression (fluorescence, growth, etc.) are omitted here (see Results),
as they do
not represent the potential functionality of our system.
Figure 2: Colony PCR after Gibson Assembly of Anderson-Promoter-Mn-Riboswitch- FAST-Phytochelatine-Terminator (GA4). This figure is an example for the high cloning efficiency when using Gibson Assembly. It also contains samples from two other composite parts (GA2 and GA3).
This composite part is a variant of
BBa_K3510003. In this composite, the phytochelatin gene is replaced
by a gene encoding a blue chromoprotein (BBa_K864401). Although this composite loses the bifunctional
characteristic of
BBa_K3510003,
it adds a mechanism for easy qualitative analysis: Color change. The
visible blue color upon expression of the chromoprotein allows qualitative in-field testing without the
necessity of fluorescence-measuring equipment. Therefore, this composite would be the cheaper
alternative of this biosensor family, and would be especially beneficial for rough examination of water
quality in areas with poor infrastructure.
Additionally, this construct was designed as a negative control for the intracellular effect of
manganese complexation by phytochelatin. The effect of phytochelatin could potentially alter the sensing
specificity of the biosensor and the viability of the host cells.
Attention: This construct does not include a ribosome binding site (RBS) upstream of the FAST2 sequence.
When building on our design, please make sure you add it in order to allow for translation.
The cloning of this composite part was successful and the steps were perfected (see Notebook)
to
increase the efficiency (Fig. 3). Due to the missing RBS in our design, the results from our
experiments
dependent on protein expression (fluorescence, color change, growth, etc.) are omitted here (see
Results), as they do not represent the potential functionality of our system.
Figure 3: Colony PCR after Gibson Assembly of Mn-Promoter-Mn-Riboswitch-FAST -Chromoprotein-Terminator (GA1). This figure is an example for the high cloning efficiency when using Gibson Assembly. It also contains samples from one other composite part (GA3).
This composite part is a variant of BBa_K3510002.
In this composite, the Mn-inducible promoter is
replaced by the Anderson promoter, a constitutive promoter. In accordance with BBa_K3510005, by removing
transcriptional regulation, direct regulation of expression occurs exclusively through the
manganese-inducible riboswitch, which regulates the translation of the mRNA. This allows a closer
analysis of the regulatory efficiency of the riboswitch and compares the functionality of the composite
with and without transcriptional regulation.
Attention: This construct does not include a ribosome binding site (RBS) upstream of the FAST2 sequence.
When building on our design, please make sure you add it in order to allow for translation.
The cloning of this composite part was successful and the steps were perfected (see Notebook)
to
increase the efficiency (Fig. 4). Due to the missing RBS in our design, the results from our
experiments
dependent on protein expression (fluorescence, color change, growth, etc.) are omitted here (see
Results), as they do not represent the potential functionality of our system.
Figure 4: Colony PCR after Gibson Assembly of Anderson-Promoter-Mn-Riboswitch-FAST -Chromoprotein-Terminator (GA3). This figure is an example for the high cloning efficiency when using Gibson Assembly. It also contains samples from one other composite part (GA1).
Vitamin B12 side project:
This composite part is a Vitamin B12-inducible expression system of a superfolder GFP (BBa_I746916). The
included part BBa_K1913008 contains a constitutive promoter (BBa_J23100) and a riboswitch. Upon binding
of Vitamin B12, the riboswitch prevents downstream translation of the transcribed gene. To express GFP
in the presence of Vitamin B12, a Tet inverter system (BBa_Q04400) is placed in
between. When the
encoded repressor is not translated anymore, the GFP is expressed. Transcriptional termination occurs
through the activity of the reliable double terminator ( BBa_B0015).
This part was inspired by Team Wageningen 2016, who provided this construct with mRFP ( BBa_K1913011). By changing the reporter protein to GFP, we aimed to provide an additional option for future iGEM teams who want to use the fluorescein-based iGEM fluorescence calibration protocol.
This part was inspired by Team Wageningen 2016, who provided this construct with mRFP ( BBa_K1913011). By changing the reporter protein to GFP, we aimed to provide an additional option for future iGEM teams who want to use the fluorescein-based iGEM fluorescence calibration protocol.
Attention: This construct does not include a ribosome binding site (RBS) upstream of the GFP sequence.
When building on our design, please make sure you add it in order to allow for translation.
This composite is a replica of iGEM team Wageningen (2016). We included it in our project as a control
of its modified version
BBa_K3510006. Moreover, our plan was to assemble the composite via Gibson
Assembly to offer a cloning alternative to the restriction-ligation method used originally.
Attention: This construct does not include a ribosome binding site (RBS) upstream of the mRFP sequence.
When building on our design, please make sure you add it in order to allow for translation.
