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Team:Tuebingen/Parts

PacMn

Parts

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).
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).

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).

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).

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).

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.
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.