Genetic design - 4C_FUELS

Cloning design

At 4C Fuels we are committed to global climate change so during last year we worked carefully to find the solution to one of the main challenges we have as humans: reducing our carbon footprint.

On this page, we are proud to present the main ideas of our cloning design, the challenges the project has gone through, and how we scientifically addressed them.

Modular Cloning: Our ASSEMBLY SYSTEM

We have assembled our genetic devices following the Modular Cloning (MoClo) framework which is based on Golden Gate Assembly Technology .

A brief reminder about Golden gate

Golden gate technology is a method which relies on the ability of Type IIS enzymes to cut outside their recognition site. The produced DNA fragments have compatible overhangs that could be efficiently assembled. One major advantage of the golden gate is the ability for the assembly of multiple DNA fragments in a single-step one-pot reaction. In addition it allows the combinatorial assembly of multiple DNA part variants.

The golden gate workflow is summarized in the following images. Briefly a mix composed of typeIIS enzymes and T4 ligase is used.

MOCLO SYSTEM

Modular Cloning or Moclo systems are a set of rules that establish a hierarchic approach for DNA Assembly. DNA constructs are divided in different assembly levels, where the DNA construction of each level poses specific features and different functionality. Likewise all DNA constructs utilized during MoClo assembly are stored in plasmids, easing their storage, cloning and exchangeability.

The jump from one level to the next one is produced when a golden gate reaction is utilized to assemble multiple previous level parts into a next level DNA construction. To do so, a set of different acceptor plasmids are used.

First of all, level 0 parts are made up of basic DNA parts such as promoters, RBS, coding sequences, or terminators. Those parts are assembled into individual Level 0 plasmids, in a process called domestication. In this way, the first reaction will result in the assembly of Level 0 parts, with a basic function. Hereinafter, Level 1 and 2 plasmids could be assembled via the combination of multiple level 0 parts into transcriptional units (Level 1 ) or multiple transcriptional units into a single construct (Level 2 ).

Several MoClo standards have emerged during the last years, each one with their specific sets of overhangs for each part type regarding its functionality. This way parts can be hierarchically assembled based on each part function. These overhang sets are also called MoClo syntaxis, where this standardized syntax allows the exchangeability of parts with other users all over the world.

Assembly Toolkit: The MARBURG COLLECTION

All of our parts follow the phytobricks syntaxis, the main standard accepted by iGEM. Moreover our assembly system is based on the design of the Marburg Collection. As many other MoClo systems, Marburg Collection is based on the usage of three sets of cloning vectors for each level 0, 1, or 2.

These acceptor vectors are unique for each level and can be utilized during three successive assembly steps to build the final construct. In this way level 0 parts are combined into level 1 plasmids and the former ones could be used to assemble a level 2. The enzyme used for assembly from Lv.1 to Lv.2 is BsaI. While for level 2 assembly Esp3I is used, which is also used for the domestication of sequences into Lv.0 parts.

Unlike other MoClo systems, the Marburg Collection features the utilization of single acceptor plasmid for each level. The hierarchical assembly of Level 2 plasmids is then achieved via the utilization of connectors.

Connectors are a new Lv.0 part which are assembled in 5´and 3´ ends of the level 1 transcriptional unit. Each connector has a recognition sequence of Esp3I, which is used for the level 2 assembly. A specific level 2 assembly overhang is placed after the Esp3I recognition sequence, which will define the position in the level 2 assembly. Thus, connectors flanking Lv.1 parts will define their final position in Lv. 2 construct.

What is more, connectors can also harbor homology regions for its integration in the microorganism’s genome.

Expanding the MoClo domestication functionality

During the design of our cloning strategy, the main problem we faced was the large amount of enzymes we needed to express in our strain.

To solve this problem, we consider grouping them into operons, allowing us to use transcriptional units where several enzymes are expressed under the control of the same regulatory elements.

The Marburg Collection design allows the assembly of operons on a level 2 using dummy promoters and terminators. However, this strategy does not allow the assembly of multiple operons in a single construct. Thus, operons are forced to be assembled under the control of a single promoter into a single level 2 transcriptional unit.

We needed to express multiple operons under the control of different promoters, and we wanted to do it in a level 2 following the standard rules of the MoClo toolbox. That is why we realized we had to come up with a solution: expanding the functionality of the level 0 domestication step.

We aim to simplify the system for polycistronic transcriptional units assembly. To do so, we have developed an expanded domestication methodology which can be helpful to other users in the community to simplify the number of steps required for complex cloning in the field of metabolic engineering.

Our domestication expansion is based on generating complex Lv. 0 parts. Unlike traditional Lv. 0 parts, our expanded parts contain more than one genetic element. For example, expanded Lv. 0 parts comprising RBS together with an enzyme or inducible promoters together with their regulation cassettes could be created.

To do so, we include additional fusion sites in each individual genetic element that is going to be assembled in the expanded Lv. 0 part. In the domestication reaction, these fusion sites join the corresponding genetic elements. Traditional Lv. 0 overhangs flank the expanded part for further Lv. 1 assembly.

Following this methodology not only a single basic element can be introduced into a level 0 part, but also new level 0 parts with extended functionality can be created. This can be achieved by the introduction of several individual elements within the universal acceptor plasmid. In addition, small sequences such as RBSs, spacers or linkers. This allows the assembly of scarless fusion proteins or polycistronic level 0 parts among others.

Nevertheless, the basic functionality of the part is preserved. That is, in the case of a CDS, it is still a coding sequence like any other level 0. Where in this case, the polycistronic CDSs allows the simultaneous expression of a complete operon instead of an isolated enzyme.

Eventually, it is important to consider that In the case of operon design, all except the first CDS element are preceded by an RBS sequence. The first CDS of the operon is not assembled with the desired RBS for the purpose of maintaining assembly standardization. This way, its RBS will be assembled during the MoClo level 1 reaction.

Pros and cons of Expanded domestication

✅ It is a simple methodology that allows to create composite zero-level parts with greater complexity without needing to reach level two to make an assembly.

✅ It not only works for assembling polycistrons into parts of Lv.0, but it also works for fusion enzymes or promoters with regulatory elements.

✅ It allows to avoid the generation of scars between the two parts that are merged. It is a seamless assembly system which can be used when the domesticated sequences are sensitive to the genetic context (introducing insulating elements flanking the composite Lv.0 part).

❌ It is an ad-hoc procedure for the specific creation of parts, requiring more design work during the domestication phase.

❌ By creating more complex core parts, the number of contexts where they could be applied are restricted, reducing the modularity of their use.

An example case of design

Our methodology is based on the design of specific domestication primers, which are applied for the primer reverse of a sequence located at 5' and the primer forward of the next enzyme located at 3'.

First of all, the primers will have a homology region with the coding sequence of the sequences to be domesticated.
In addition, in order to add a different RBS to the one that exists in the previous sequence, the primer forward will include in 5' the RBS sequence that you want to include in the new polycistron variant.
Finally, both the first forward and the reverse, will then include a unique overhang, specific and different from the rest of the overhangs used during the domestication reaction. In our case, this homology region has been defined as “AACG.” However, the last 4 bases of the RBS will include as part of a polycistron, a linker between enzymes or any other short sequence to be introduced that can be used as an overhang, being necessary to include them in this case also in the primer reverse 3`.

Similarly, the primer forward of the enzyme located at 5' and the primer reverse of the enzyme located at 3', have to carry the standard overhangs of domestication. As well as another 4 extra bp, specific to the type of part, which will allow their correct assembly as parts of Lv.1 at a later date. In the particular case of the CDS of the Marburg Collection, the primer would have the overhangs ' “AACGTCTCGCTCGAATG” at 5` and “TT CGT CTC CCT CAA AGC” at 3`.

After the PCR assembly of both enzymes with a new RBS located between them, they are finally assembled into a part of Lv.0 via golden gate reaction. In-silico designs are depicted in the image below.

Expanding the MoClo domestication functionality example: As can be observed, the in-silico designed RBS “RBS_PhaB^173S” ( BBa_K3726006 ) is going to be replaced by RBS* ( BBa_K3726093 ) with the objective of creating new variants of this polycistronic sequence with different expresión levels.

We have also used this methodology for the creation of scarless fusion enzymes. First, stop codons from the upstream coding sequence are eliminated during the PCR domestication, and a small linker is codified in the domestication primer, in order to fuse both proteins. To know more about why creating fusion enzymes, visit the Metabollic Engineering Page. Using this methodology, the following parts were constructed: CDS_Lv0_BOH1_C_GSG ( BBa_K3726017 ) and CDS_Lv0_BOH1_C_FRRRF ( BBa_K3726018 ).

Our Expression Constructs

After selecting our cloning methodology and thoughtfully defining our metabolic Engineering strategy, we planned the design of our genetic constructs. To achieve the required aims for pathway optimization, we decided to create multiple variants of each construct, evalute them combinatorially and eventually selecting the best performing combinations.

The General design strategy

In the beginning of the project we performed intensive research to select and design our regulatory elements such as promoters, RBS and terminators, as well as our coding sequences and other required elements. In addition, we were kindly provided with a set of useful parts from the Marburg iGEM Team.

During this process, we discovered that RBS and mRNA stability in cyanobacteria pose a highly relevant role in expression regulation.Then we focused our design in the efficient grouping of our coding sequences into carefully designed operons. To achieve this task, we have supported our design work with the help of DeNovoDNA Software Tools, who kindly sponsored our iGEM team.

This way, all the genetic design and sequences you will find below have been performed considering the optimal arrangement of enzymes into artificial operons, as well as the overall mRNA stability. High or low expression requirements of constructs are either achieved by promoters and the utilization of different strength in-silico designed RBS elements, which specifically account for the influence of the surrounding genetic context. In addition, sequences have been codon optimized for Synechococcus elongatus PCC7942 and internal RNAse recognition sites have been removed to improve mRNA stability.

In the end, all of these efforts translated into the successful assembly of more than 30 level 0 parts, among them multiple strong constitutive promoters for cyanobacteria, a specific highly efficient rho-independent terminator or selective n-butanol export pump. To explore further all of these parts, visit our Parts collection page.

Expression Devices

Once we define our level 0 parts toolbox, we move forward to plan our genetic constructs. Click the buttons below to know more about each one of our designed expression constructs. For the assembly of our devices we have employed 3 different acceptor plasmids.

Level 1 assemblies were performed within the Level 1 AmpR / ColE1 RFP Dropout acceptor for all the devices except Tolerance & secretion constructs, which were assembled within the Level 1 SpecR / ColE1+pANs entry vector, a shuttle vector with the pANs origin for replication within Synechococcus elongatus PCC7942 and related organisms species.

Level 2 assemblies were performed within the Level 2 KanR / ColE1 / RFP Dropout acceptor .

Explore our designed expression constructs

L1_BOH3-A

5CON1-NSI

Prbcl-m36

RBS NphT7

BOH3

Bba_0015 (5a)

KanR

3CON5-NSI

This construct is an integrative plasmid, harboring a transcriptional unit for the expression of an oxygen-tolerant butanol biosynthesis pathway.

L1_BOH3-A corresponds with the composite part “ BBa_K3726044” It is a transcriptional unit for the expression of the part “ BBa_K3726011 " CDS_Lv0_BOH_3 which is a MoClo Lv.0 CDS element.

CDS_Lv0_BOH_3 is a polycistronic CDS containing the six enzymes for the synthetic n-butanol biosynthesis pathway. Those enzymes are: CDS_NphT7 ( BBa_K3726000), CDS_PhaB^173S ( BBa_K3726001 ), CDS_Ccr ( BBa_K3726002 ), CDS_PhaJ ( BBa_K3726003 ), CDS_PduP ( BBa_K3726004 ), CDS_Slr1192 ( BBa_K3726005 ). Within this artificial operon each CDS element is preceded by an in-silico designed RBS sequence using DeNovo DNA software.

Expression is driven by the strong constitutive promoter Prbcl-m36 “ BBa_K3726080 ” and the strong synthetic RBS, RBS_NphT7 “ BBa_K3726088”, as well as the internal RBS present within the polycistronic CDS.

The relative transcription initiation strengths of each RBS in the operon context has been calculated with DeNovo DNA Software. Results are depicted in the graphic below.

As can be observed, the PduP and Slr1192 internal RBS sequences have been designed to pose a moderate strength. Since this part corresponds with a fully functional n-butanol biosynthesis, relative expression of each enzyme required for optimal pathway functioning is controlled by their internal RBS.

To achieve the integration of the expression cassette via double homologous recombination within the genome. L1_BOH3-A is flanked by two homology regions “ BBa_K3726104” 5CON1(H)_NS1(mod)-up (PCC 11801 ) and “ BBa_K3726105 ” 3CON5(H)_NS1(mod)-down (PCC 11801 ). Likewise, a Kanamycin resistance cassette is included downstream the transcriptional unit region.

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L1_BOH3-B