Team:MADRID UCM/SynbioDesign

Genetic design - 4C_FUELS

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.

Golden Gate Image 1
Golden Gate Image 1
Golden Gate Image 1

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 ).

DESCRIPCIΓ“N

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.

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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 5Β΄and 3Β΄ 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.

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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.

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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

βœ… 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.

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

βœ… 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).

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❌ 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 β€œAACG.” 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 ' β€œAACGTCTCGCTCGAATG” at 5` and β€œTT 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.

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Expanding the MoClo domestication functionality example: As can be observed, the in-silico designed RBS β€œRBS_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 expresiΓ³n 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

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5CON1-NSI
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Prbcl-m36
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RBS NphT7
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BOH3
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Bba_0015 (5a)
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KanR
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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 β€œ BBa_K3726044” It is a transcriptional unit for the expression of the part β€œ 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 β€œ BBa_K3726080 ” and the strong synthetic RBS, RBS_NphT7 β€œ 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.

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To achieve the integration of the expression cassette via double homologous recombination within the genome. L1_BOH3-A is flanked by two homology regions β€œ BBa_K3726104” 5CON1(H)_NS1(mod)-up (PCC 11801 ) and β€œ 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

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5CON1-NSI
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J23119
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RBS NphT7
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BOH3
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Bba_0015 (5a)
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KanR
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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 β€œ BBa_K3726044” It is a transcriptional unit for the expression of the part β€œ 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 J23119 β€œ BBa_K2560031 " and the strong synthetic RBS, RBS_NphT7 β€œ 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.

Alt

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 β€œ BBa_K3726104” 5CON1(H)_NS1(mod)-up (PCC 11801 ) and β€œ 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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L2_BOH1ABC

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5CON1-NS1
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TU_BOH_1A
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TU_BOH_1B
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TU_BOH_1C (KanR)
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3CON5-NS1

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

L2_BOH1ABC corresponds with the composite part β€œ BBa_K3726058” It is a level 2 MoClo part which has three transcriptional units for the expression of the MoClo Lv.1 parts β€œ BBa_K3726044” BOH1_A_LV1, β€œ BBa_K3726045” BOH1_B_LV1 and β€œ BBa_K3726046” BOH1_C_LV1.

BOH1_A_LV1, BOH1_B_LV1 and BOH1_C_LV1 are transcriptional units for the expression of the parts β€œ BBa_K3726012 " CDS_Lv0_BOH_1_A , β€œ BBa_K3726013 " CDS_Lv0_BOH_1_B and β€œ BBa_K3726014” CDS_Lv0_BOH_1_C which are a MoClo Lv.0 CDS elements.

CDS_Lv0_BOH_1_A, is an artificial polycistronic CDS designed to express the first two enzymes for the synthetic n-butanol biosynthesis pathway.Those enzymes are: CDS_NphT7 ( BBa_K3726000), CDS_PhaB^173S ( BBa_K3726001 ). CDS_Lv0_BOH_1_B is another artificial polycistronic CDS designed to express the third and the fourth enzymes, which are: CDS_Ccr ( BBa_K3726002 ) and CDS_PhaJ ( BBa_K3726003 ). And for last, CDS_Lv0_BOH_1_C is another artificial polycistronic CDS designed to express the last two enzymes, which are: CDS_PduP ( BBa_K3726004 ) and CDS_Slr1192 ( BBa_K3726005 ).

For BOH1_A_LV1, the expression is driven by the strong constitutive promoter , RBS* β€œ BBa_K3726093 ", which has demonstrated its increased ribosome binding strength in Synechocystis PCC6803 and other cyanobacteria in comparison with other heterotroph-derived RBS . As well, the expression is also driven by the internal RBS presented within the polycistronic CDS.

For BOH1_B_LV1, expression is driven by the strong constitutive promoter Prbcl-m36 β€œ BBa_K3726080 ”and by the strong synthetic RBS , RBS_BCD2(mod) β€œ BBa_K3726086”, as well as the internal RBS presented within the polycistronic CDS.

And for BOH1_C_LV1, expression is driven by the medium-strong synthetic constitutive promoter J23119 β€œ BBa_K2560031 "and by the synthetic RBS, RBS* β€œ BBa_K3726093 ", as well as the internal RBS presented within the polycistronic CDS.

To achieve the integration of the expression cassette via double homologous recombination within the genome. L2_BOH1ABC is flanked by two homology regions β€œ BBa_K3726104” 5CON1(H)_NS1(mod)-up (PCC 11801 ) and β€œ BBa_K3726105 ” 3CON5(H)_NS1(mod)-down (PCC 11801 ). Likewise, a Kanamycin resistance cassette is included downstream the transcriptional unit region β€œLv1_BOH1_C”.

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L2_BOH2AB

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5CON1-NS1
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TU_BOH_2A
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TU_BOH_2B (KanR)
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3CON5-NS1

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

L2_BOH2AB corresponds with the composite part β€œ BBa_K3726057” It is a level 2 MoClo part which has two transcriptional units for the expression of the MoClo Lv.1

parts β€œ BBa_K3726047” BOH2_A_LV1 and β€œ BBa_K3726048” BOH2_B_LV1.

BOH2_A_LV1 and BOH2_B_LV1 are transcriptional units for the expression of the parts β€œ BBa_K3726015” CDS_Lv0_BOH_2_A and β€œ BBa_K3726016” CDS_Lv0_BOH_2_B which are a MoClo Lv.0 CDS elements.

CDS_Lv0_BOH_2_A, is an artificial polycistronic CDS designed to express the first four 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 ), and CDS_Lv0_BOH_2_B is another artificial polycistronic CDS designed to express the last two, which are: CDS_PduP ( BBa_K3726004 ) and CDS_Slr1192 ( BBa_K3726005 ). Within these artificial operons each CDS element is preceded by an in-silico designed RBS sequence using DeNovo DNA software.

For BOH2_A_LV1, expression is driven by the strong constitutive promoter P_PpsbA*-RiboJ β€œ BBa_K3726079” and the strong synthetic RBS, RBS_NphT7 β€œ BBa_K3726088” , as well as the internal RBS presented within the polycistronic CDS.

And for BOH2_B_LV1, expression is driven by the medium-strong synthetic constitutive promoter J23119 β€œ BBa_K2560031 "and by the strong synthetic RBS, RBS_PduP β€œ BBa_K3726090” , as well as the internal RBS presented 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.

For BOH2A

Internal RBS sequences are conserved from BOH3 part ( BBa_K3726011 ). Transcription initiation rate is high for the encoded enzymes.


For BOH2B

The designed RBS of this operon has been changed from BOH3 part ( BBa_K3726011 ).RBS sequences have been recalculated to pose a higher strength.



In the case of BOH2B, the part encodes the two last enzymes of the operon whose relative expression level should be lower than the other enzymes. The designed RBS of this operon has been changed from BOH3 part ( BBa_K3726011 ), since expression can now be controlled via the promoter element during the assembly of this part within a transcriptional unit. Then, the RBS sequences have been recalculated to pose a higher strength.

To achieve the integration of the expression cassette via double homologous recombination within the genome. L2_BOH2AB is flanked by two homology regions β€œ BBa_K3726104” 5CON1(H)_NS1(mod)-up (PCC 11801 ) and β€œ BBa_K3726105 ” 3CON5(H)_NS1(mod)-down (PCC 11801 ). Likewise, a Kanamycin resistance cassette is included downstream the transcriptional unit region β€œLv1_BOH2_B”.

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L2_BOH2AB-GSG

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5CON1-NS1
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TU_BOH_2A
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TU_BOH_2B-GSG (KanR)
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3CON5-NS1

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

L2_BOH2A-GSG corresponds with the composite part β€œ BBa_K3726060” It is a level 2 MoClo part which has two transcriptional units for the expression of the MoClo Lv.1 parts β€œ BBa_K3726047” BOH2_A_LV1 and β€œ BBa_K3726052 " BOH1_C_GSG_LV1.

BOH2_A_LV1 and BOH1_C_GSG_LV1 are transcriptional units for the expression of the parts β€œ BBa_K3726015” CDS_Lv0_BOH_2_A and β€œ BBa_K3726017” CDS_Lv0_BOH_1_C_GSG which are a MoClo Lv.0 CDS elements.

CDS_Lv0_BOH_2_A, is an artificial polycistronic CDS designed to express the first four 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 ), and CDS_Lv0_BOH_1_C_GSG is another artificial polycistronic CDS designed to express the last two, which are: CDS_PduP ( BBa_K3726004 ) and CDS_Slr1192 ( BBa_K3726005 ). The protein encoded by this part corresponds with a chimeric protein, created by the fusion of two different enzymes using a linker. Within the Lv0_BOH_2_A operon, each CDS element is preceded by an in-silico designed RBS sequence using DeNovo DNA software.

For BOH2_A_LV1, expression is driven by the strong constitutive promoter P_PpsbA*-RiboJ β€œ BBa_K3726079” and the strong synthetic RBS, RBS_NphT7 β€œ BBa_K3726088” , as well as the internal RBS presented within the polycistronic CDS.

And for BOH1_C_GSG_LV1, expression is driven by the medium-strong synthetic constitutive promoter J23119 β€œ BBa_K2560031 "and by the strong synthetic RBS, RBS_PduP β€œ BBa_K3726090”, as well as the internal RBS presented within the polycistronic CDS.

The relative transcription initiation strengths of each RBS in BOH2_A_LV1 operon context has been calculated with DeNovo DNA Software. Results are depicted in the graphic below.

Internal RBS sequences are conserved from BOH3 part ( BBa_K3726011 ). Transcription initiation rate is high for the encoded enzymes.

For BOH2A
Alt

To achieve the integration of the expression cassette via double homologous recombination within the genome. L2_BOH2A-GSG is flanked by two homology regions β€œ BBa_K3726104” 5CON1(H)_NS1(mod)-up (PCC 11801 ) and β€œ BBa_K3726105 ” 3CON5(H)_NS1(mod)-down (PCC 11801 ). Likewise, a Kanamycin resistance cassette is included downstream the transcriptional unit region β€œBOH1_C_GSG_LV1 ".

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L2_BOH2AB-FR3F

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5CON1-NS1
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TU_BOH_2A
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TU_BOH_2B-FR3F (KanR)
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3CON5-NS1

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

L2_BOH2A-FRRRF corresponds with the composite part β€œ BBa_K3726059” It is a level 2 MoClo part which has two transcriptional units for the expression of the MoClo Lv.1 parts β€œ BBa_K3726047” BOH2_A_LV1 and β€œ BBa_K3726051 " BOH1_C_FRRRF_LV1.

BOH2_A_LV1 and BOH1_C_FRRRF_LV1 are transcriptional units for the expression of the parts β€œ BBa_K3726015” CDS_Lv0_BOH_2_A and β€œ BBa_K3726018” CDS_Lv0_BOH_1_C_FRRRF which are a MoClo Lv.0 CDS elements.

CDS_Lv0_BOH_2_A, is an artificial polycistronic CDS designed to express the first four 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 ), and CDS_Lv0_BOH_1_C_FRRRF is another artificial polycistronic CDS designed to express the last two, which are: CDS_PduP ( BBa_K3726004 ) and CDS_Slr1192 ( BBa_K3726005 ). The protein encoded by this part corresponds with a chimeric protein, created by the fusion of two different enzymes using a linker. Within the BOH2_A_LV1 l operon, each CDS element is preceded by an in-silico designed RBS sequence using DeNovo DNA software.

For BOH2_A_LV1, expression is driven by the strong constitutive promoter P_PpsbA*-RiboJ β€œ BBa_K3726079” and the strong synthetic RBS, RBS_NphT7 β€œ BBa_K3726088” , as well as the internal RBS presented within the polycistronic CDS.

And for BOH1_C_FRRRF_LV1, expression is driven by the medium-strong synthetic constitutive promoter J23119 β€œ BBa_K2560031 "and by the strong synthetic RBS, RBS_PduP β€œ BBa_K3726090”, as well as the internal RBS presented within the polycistronic CDS.

The relative transcription initiation strengths of each RBS in BOH2_A_LV1 operon context has been calculated with DeNovo DNA Software. Results are depicted in the graphic below.

Internal RBS sequences are conserved from BOH3 part ( BBa_K3726011 ). Transcription initiation rate is high for the encoded enzymes.

For BOH2A
Alt

To achieve the integration of the expression cassette via double homologous recombination within the genome. L2_BOH2A-GSG is flanked by two homology regions β€œ BBa_K3726104” 5CON1(H)_NS1(mod)-up (PCC 11801 ) and β€œ BBa_K3726105 ” 3CON5(H)_NS1(mod)-down (PCC 11801 ). Likewise, a Kanamycin resistance cassette is included downstream the transcriptional unit region β€œBOH1_C_FRRRF_LV1 ".

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L2_BOH1ABC-GSG

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5CON1-NS1
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TU_BOH_1A
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TU_BOH_1B
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TU_BOH_2B-GSG (KanR)
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3CON5-NS1

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

L2_BOH1ABC-GSG corresponds with the composite part β€œ BBa_K3726060” It is a level 2 MoClo part which has three transcriptional units for the expression of the MoClo Lv.1 parts β€œ BBa_K3726044” BOH1_A_LV1, β€œ BBa_K3726045” BOH1_B_LV1 and β€œ BBa_K3726052 " BOH1_C_GSG_LV1.

BOH1_A_LV1, BOH1_B_LV1 and BOH1_C_GSG_LV1 are transcriptional units for the expression of the parts β€œ BBa_K3726012 " CDS_Lv0_BOH_1_A , β€œ BBa_K3726013 " CDS_Lv0_BOH_1_B and β€œ BBa_K3726017” CDS_Lv0_BOH_1_C_GSG which are a MoClo Lv.0 CDS elements.

CDS_Lv0_BOH_1_A, is an artificial polycistronic CDS designed to express the first two enzymes for the synthetic n-butanol biosynthesis pathway.Those enzymes are: CDS_NphT7 ( BBa_K3726000), CDS_PhaB^173S ( BBa_K3726001 ). CDS_Lv0_BOH_1_B is another artificial polycistronic CDS designed to express the third and the fourth enzymes, which are: CDS_Ccr ( BBa_K3726002 ) and CDS_PhaJ ( BBa_K3726003 ). And for last, CDS_Lv0_BOH_1_C_GSG is another artificial polycistronic CDS designed to express the last two enzymes, which are: CDS_PduP ( BBa_K3726004 ) and CDS_Slr1192 ( BBa_K3726005 ). The protein encoded by this part corresponds with a chimeric protein, created by the fusion of two different enzymes using a linker.

For BOH1_A_LV1, the expression is driven by the strong constitutive promoter , RBS* β€œ BBa_K3726093 ", which has demonstrated its increased ribosome binding strength in Synechocystis PCC6803 and other cyanobacteria in comparison with other heterotroph-derived RBS . As well, the expression is also driven by the internal RBS presented within the polycistronic CDS.

For BOH1_B_LV1, expression is driven by the strong constitutive promoter Prbcl-m36 β€œ BBa_K3726080 ”and by the strong synthetic RBS , RBS_BCD2(mod) β€œ BBa_K3726086”, as well as the internal RBS presented within the polycistronic CDS.

And for BOH1_C_GSG_LV1, expression is driven by the medium-strong synthetic constitutive promoter J23119 β€œ BBa_K2560031 "and by the strong synthetic RBS, RBS_PduP β€œ BBa_K3726090”, as well as the internal RBS presented within the polycistronic CDS.

To achieve the integration of the expression cassette via double homologous recombination within the genome. L2_BOH1ABC is flanked by two homology regions β€œ BBa_K3726104” 5CON1(H)_NS1(mod)-up (PCC 11801 ) and β€œ BBa_K3726105 ” 3CON5(H)_NS1(mod)-down (PCC 11801 ). Likewise, a Kanamycin resistance cassette is included downstream the transcriptional unit region β€œLv1_BOH1_C_GSG”.

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L2_BOH1ABC-FR3F

...
5CON1-NS1
...
TU_BOH_1A
...
TU_BOH_1B
...
TU_BOH_2B-FR3F (KanR)
...
3CON5-NS1

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

L2_BOH1ABC-FRRRF corresponds with the composite part β€œ BBa_K3726059” It is a level 2 MoClo part which has three transcriptional units for the expression of the MoClo Lv.1 parts β€œ BBa_K3726044” BOH1_A_LV1, β€œ BBa_K3726045” BOH1_B_LV1 and β€œ BBa_K3726051 " BOH1_C_FRRRF_LV1.

BOH1_A_LV1, BOH1_B_LV1 and BOH1_C_FRRRF_LV1 are transcriptional units for the expression of the parts β€œ BBa_K3726012 " CDS_Lv0_BOH_1_A , β€œ BBa_K3726013 " CDS_Lv0_BOH_1_B and β€œ BBa_K3726018” CDS_Lv0_BOH_1_C_FRRRF which are a MoClo Lv.0 CDS elements.

CDS_Lv0_BOH_1_A, is an artificial polycistronic CDS designed to express the first two enzymes for the synthetic n-butanol biosynthesis pathway.Those enzymes are: CDS_NphT7 ( BBa_K3726000), CDS_PhaB^173S ( BBa_K3726001 ). CDS_Lv0_BOH_1_B is another artificial polycistronic CDS designed to express the third and the fourth enzymes, which are: CDS_Ccr ( BBa_K3726002 ) and CDS_PhaJ ( BBa_K3726003 ). And for last, CDS_Lv0_BOH_1_C_FRRRF is another artificial polycistronic CDS designed to express the last two enzymes, which are: CDS_PduP ( BBa_K3726004 ) and CDS_Slr1192 ( BBa_K3726005 ). The protein encoded by this part corresponds with a chimeric protein, created by the fusion of two different enzymes using a linker.

For BOH1_A_LV1, the expression is driven by the strong constitutive promoter , RBS* β€œ BBa_K3726093 ", which has demonstrated its increased ribosome binding strength in Synechocystis PCC6803 and other cyanobacteria in comparison with other heterotroph-derived RBS . As well, the expression is also driven by the internal RBS presented within the polycistronic CDS.

For BOH1_B_LV1, expression is driven by the strong constitutive promoter Prbcl-m36 β€œ BBa_K3726080 ”and by the strong synthetic RBS , RBS_BCD2(mod) β€œ BBa_K3726086”, as well as the internal RBS presented within the polycistronic CDS

And for BOH1_C_FRRRF_LV1, expression is driven by the medium-strong synthetic constitutive promoter J23119 β€œ BBa_K2560031 "and by the strong synthetic RBS, RBS_PduP β€œ BBa_K3726090”, as well as the internal RBS presented within the polycistronic CDS.

To achieve the integration of the expression cassette via double homologous recombination within the genome. L2_BOH1ABC is flanked by two homology regions β€œ BBa_K3726104” 5CON1(H)_NS1(mod)-up (PCC 11801 ) and β€œ BBa_K3726105 ” 3CON5(H)_NS1(mod)-down (PCC 11801 ). Likewise, a Kanamycin resistance cassette is included downstream the transcriptional unit region β€œLv1_BOH1_C_FRRRF”.

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L1_HspA-A

...
5CON1-NSIII
...
PpsbA*-RiboJ
...
RBS PtaBs
...
HspA(11801 )
...
Bba_0015 (5a)
...
CmlR
...
3CON1

This construct is assembled within a shuttle plasmid, harboring the pANs cyanobacterial origin of replication ( BBa_K3228069 )It is designed for the expression of a transcriptional unit, codifying for the major heat shock protein capable of enhancing cellular fitness in the presence of environmental stresses such as solvent presence. Within our project we have used HspA to reduce ROS stress generated from n-butanol.

L1_HspA-A corresponds with the composite part β€œ BBa_K3726066” It is a transcriptional unit for the expression of the part β€œ BBa_K3726061 " CDS_Lv0_HspA* which is a MoClo Lv.0 CDS element.

Expression is driven by the strong constitutive promoter P_PpsbA*-RiboJ β€œ BBa_K3726079” and the strong synthetic RBS, RBS_PtaBs β€œ BBa_K3726092 ".

The part in its upstream region has a homology region β€œ BBa_K3726108” 5CON1(H)_NS3(mod)-up (PCC 11801 ) for homologous recombination within the genome of PCC 11801. In the downstream region, the part has the connector 3'Con1 β€œ BBa_K2560070” that will allow the assembly of a Lv2 construct following the marburg collection standard. This design will allow us to test which β€œtolerance and secretion ” variant has better efficiency by its own account as a replicative vector, and then assemble them as an integrative Lv2 Moclo construct.

For last, a chloramphenicol resistance cassette is included downstream the transcriptional unit region.

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L1_HspA-B

...
5CON1-NSIII
...
PS-PR
...
RBS PtaBs
...
HspA(11801 )
...
Bba_0015 (5a)
...
CmlR
...
3CON1

TThis construct is assembled within a shuttle plasmid, harboring the pANs cyanobacterial origin of replication ( BBa_K3228069 )It is designed for the expression of a transcriptional unit, codifying for the major heat shock protein capable of enhancing cellular fitness in the presence of environmental stresses such as solvent presence. Within our project we have used HspA to reduce ROS stress generated from n-butanol.

L1_HspA-B corresponds with the composite part β€œ BBa_K3726067” It is a transcriptional unit for the expression of the part β€œ BBa_K3726061 " CDS_Lv0_HspA* which is a MoClo Lv.0 CDS element.

Expression is driven by the strong constitutive promoter for cyanobacteria, PS-PR β€œ BBa_K3726084” and the strong synthetic RBS, RBS_PtaBs β€œ BBa_K3726092 ".

The part in its upstream region has a homology region β€œ BBa_K3726108” 5CON1(H)_NS3(mod)-up (PCC 11801 ) for homologous recombination within the genome of PCC 11801. In the downstream region, the part has the connector 3'Con1 β€œ BBa_K2560070” that will allow the assembly of a Lv2 construct following the marburg collection standard. This design will allow us to test which β€œtolerance and secretion ” variant has better efficiency by its own account as a replicative vector, and then assemble them as an integrative Lv2 Moclo construct.

For last, a chloramphenicol resistance cassette is included downstream the transcriptional unit region.

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L1_SigB-A

...
5CON3
...
Prbcl-m36
...
RBS PtaBs
...
SigB(11801 )
...
Teck (5 )
...
3CON5-NSIII

This construct is assembled within a shuttle plasmid, harboring the pANs cyanobacterial origin of replication ( BBa_K3228069 )It is designed for the expression of a transcriptional unit, codifying for a sigma factor that when it is over-expressed increases tolerance to temperature, and reduces ROS stress. Within our project we have used SigB to reduce ROS stress generated from n-butanol.

L1_SigB-A corresponds to the composite part β€œ BBa_K3726068”. It is a transcriptional unit for the expression of the part β€œ BBa_K3726063 " CDS_Lv0_SigB* which is a MoClo Lv.0 CDS element.

Expression is driven by the strong constitutive promoter Prbcl-m36 β€œ BBa_K3726080” and the strong synthetic RBS, RBS_PtaBs β€œ BBa_K3726092 ".

The part in its upstream region, has the connector 5'Con3 β€œK2560067” that will allow the assembly of a Lv2 construct following the marburg collection standard. In its downstream region has a homology region β€œ BBa_K3726105” 3CON5(H)_NS1(mod)-down (PCC 11801 ) for homologous recombination within the genome of PCC 11801. This design will allow us to test which β€œtolerance and secretion ” variant has better efficiency by its own account as a replicative vector, and then assemble them as an integrative Lv2 Moclo construct.

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L1_SigB-B

...
5CON3
...
J23119
...
RBS PtaBs
...
SigB(11801 )
...
Teck (5 )
...
3CON5-NSIII

This construct is assembled within a shuttle plasmid, harboring the pANs cyanobacterial origin of replication ( BBa_K3228069 )It is designed for the expression of a transcriptional unit, codifying for a sigma factor that when it is over-expressed increases tolerance to temperature, and reduces ROS stress. Within our project we have used SigB to reduce ROS stress generated from n-butanol.

L1_SigB-B corresponds to the composite part β€œ BBa_K3726069”. It is a transcriptional unit for the expression of the part β€œ BBa_K3726063 " CDS_Lv0_SigB* which is a MoClo Lv.0 CDS element.

Expression is driven by the medium-strong synthetic constitutive promoter J23119 β€œ BBa_K2560031 "and the strong synthetic RBS, RBS_PtaBs β€œ BBa_K3726092 ".

The part in its upstream region, has the connector 5'Con3 β€œK2560067” that will allow the assembly of a Lv2 construct following the marburg collection standard. In its downstream region has a homology region β€œ BBa_K3726109” 3CON5(H)_NS3(mod)-down (PCC 11801 ) for homologous recombination within the genome of PCC 11801. This design will allow us to test which β€œtolerance and secretion ” variant has better efficiency by its own account as a replicative vector, and then assemble them as an integrative Lv2 Moclo construct.

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L2_Tolerance (pANs)

...
5CON1-NSIII
...
TU_HspA-X (CmlR)
...
TU_AcrbV2-X
...
TU_SigB-X
...
3CON5-NSIII

This construct is an integrative plasmid, harboring a transcriptional unit for the expression of all β€œtolerance and secretion” constructs within PCC 11801 Genome. It is a system that combines the simultaneous effect of all the parts, to study their influence when combining the effects and separately.

L2_TOL-Mix corresponds with the composite part β€œ BBa_K3726073 " It is a level 2 MoClo part which has three transcriptional units for the expression of the MoClo Lv.1 parts β€œ BBa_K3726066” Lv1_ HspA-A, β€œ BBa_K3726071 " Lv1_AcrbV2 - B and β€œ BBa_K3726069” Lv1 SigB-B.

Lv1_ HspA-A, Lv1_AcrbV2 - B and Lv1 SigB-B are transcriptional units for the expression of the parts β€œ BBa_K3726061 " CDS_Lv0_HspA* , β€œ BBa_K3726065” CDS_Lv0_AcrbV2 and β€œ BBa_K3726063 " CDS_Lv0_SigB* which are MoClo Lv.0 CDS elements.

This part has been assembled following the marburg collection standard, then the transcriptional units are flanked by two homology regions β€œ BBa_K3726108” 5CON1(H)_NS3(mod)-up (PCC 11801 ) and β€œ BBa_K3726109” 3CON5(H)_NS3(mod)-down (PCC 11801 ) that allows the double homologous recombination within the genome of PCC 11801.

For last, a chloramphenicol resistance cassette is included downstream Lv1_ HspA-A transcriptional unit region.

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L1_AcrbV2-A

...
5CON2
...
AraC+pBAD
...
RBS PtaBs
...
AcrbV2
...
Teck (5 )
...
3CON2

This construct is assembled within a shuttle plasmid, harboring the pANs cyanobacterial origin of replication ( BBa_K3228069 )It is designed for the expression of a transcriptional unit, codifying for a membrane transporter subunit, which has been optimized for the specific secretion of n-butanol. Within our project we have used AcrbV2 to enhance n-butanol secretion through cell membrane in order to cut down on toxicity generated by itself.

L1_AcrbV2-A corresponds to the composite part β€œ BBa_K3726070”. It is a transcriptional unit for the expression of the part β€œ BBa_K3726065” CDS_Lv0_AcrbV2 which is a MoClo Lv.0 CDS element.

Expression is driven by the Medium-Strong inducible promoter pBAD β€œ BBa_K3726076” which is regulated by the presence of Arabinose, and the strong synthetic RBS, RBS_PtaBs β€œ BBa_K3726092 ".

The part is flanked by two connectors that will allow the assembly of a Lv2 construct following the marburg collection standard. In its upstream region the connector is 5'Con2 Bba_K2560066, and in its downstream region 3'Con2 Bba_K2560071. This design will allow us to test which β€œtolerance and secretion ” variant has better efficiency by its own account as a replicative vector, and then assemble them as an integrative Lv2 Moclo construct.

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L1_AcrbV2-Bh

...
5CON2
...
Pgntk
...
RBS PtaBs
...
AcrbV2
...
Teck (5 )
...
3CON2

This construct is assembled within a shuttle plasmid, harboring the pANs cyanobacterial origin of replication ( BBa_K3228069 )It is designed for the expression of a transcriptional unit, codifying for a membrane transporter subunit, which has been optimized for the specific secretion of n-butanol. Within our project we have used AcrbV2 to enhance n-butanol secretion through cell membrane in order to cut down on toxicity generated by itself.

L1_AcrbV2-B corresponds to the composite part β€œ BBa_K3726071 ". It is a transcriptional unit for the expression of the part β€œ BBa_K3726065” CDS_Lv0_AcrbV2 which is a MoClo Lv.0 CDS element.

Expression is driven by the promoter PgntK β€œ BBa_K3726078” which is a negative feedback membrane stress promoter that has demonstrated that can optimize the expression levels of AcrB drug efflux pump to maximize the growth rate and minimize cellular toxicity. Moreover, the expression is controlled as well by the strong synthetic RBS, RBS_PtaBs β€œ BBa_K3726092 ".

The part is flanked by two connectors that will allow the assembly of a Lv2 construct following the marburg collection standard. In its upstream region the connector is 5'Con2 Bba_K2560066, and in its downstream region 3'Con2 Bba_K2560071. This design will allow us to test which β€œtolerance and secretion ” variant has better efficiency by its own account as a replicative vector, and then assemble them as an integrative Lv2 Moclo construct.

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L1_AcrbV2-C

...
5CON2
...
J23119
...
RBS BCD2*(mod)
...
AcrbV2
...
Teck (5 )
...
3CON2

This construct is assembled within a shuttle plasmid, harboring the pANs cyanobacterial origin of replication ( BBa_K3228069 )It is designed for the expression of a transcriptional unit, codifying for a membrane transporter subunit, which has been optimized for the specific secretion of n-butanol. Within our project we have used AcrbV2 to enhance n-butanol secretion through cell membrane in order to cut down on toxicity generated by itself.

L1_AcrbV2-C corresponds to the composite part β€œ BBa_K3726072 ". It is a transcriptional unit for the expression of the part β€œ BBa_K3726065” CDS_Lv0_AcrbV2 which is a MoClo Lv.0 CDS element.

Expression is driven by the medium-strong synthetic constitutive promoter J23119 β€œ BBa_K2560031 " and by the strong synthetic RBS, BCD2(mod) β€œ BBa_K3726086”.

The part is flanked by two connectors that will allow the assembly of a Lv2 construct following the marburg collection standard. In its upstream region the connector is 5'Con2 Bba_K2560066, and in its downstream region 3'Con2 Bba_K2560071. This design will allow us to test which β€œtolerance and secretion ” variant has better efficiency by its own account as a replicative vector, and then assemble them as an integrative Lv2 Moclo construct.

L2_TOL_ABB
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L1_PKMCG-A

...
5CON1-NSV
...
PpsbA*-RiboJ
...
RBS PtaBs
...
PKMCG
...
Bba_0015 (5a)
...
SpecR
...
3CON5-NSV

This construct is an integrative plasmid, harboring a transcriptional unit for the expression of the half part of the MCG cycle and the enzymes that belong to the phosphoketolase pathway. If you want to know more about the MCG cycle or phosphoketolase pathway, visit the Metabolic Engineering Page.

L1-PKMCG-A, corresponds with the composite part β€œ BBa_K3726053 " It is a transcriptional unit for the expression of the part β€œ BBa_K3726028” CDS_Lv0_MCG-PK which is a MoClo Lv.0 CDS element.

CDS_Lv0_MCG-PK is a polycistronic CDS that contains the half part of the enzymes of the MCG cycle and the enzymes that belong to the phosphoketolase pathway. Those enzymes are: CDS_PtaBs ( BBa_K3726019 ), CDS_PKPa ( BBa_K3726021 ), CDS_mdh ( BBa_K3726023 ), CDS_GarK ( BBa_K3726025 ), garR_CDS ( BBa_K3726027 ). 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 P_PpsbA*-RiboJ β€œ BBa_K3726079” and the strong synthetic RBS, RBS_PtaBs β€œ BBa_K3726092 " , as well as the internal RBS presented within the polycistronic CDS.

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

Alt

Since this part corresponds with the half part of a fully functional artificial carbon fixation pathway & bypass towards acetyl-CoA formation, relative expression of each enzyme required for optimal pathway functioning is controlled by their internal RBS.

As can be observed, the PtaBs initial RBS sequence have been designed to pose a moderate strength. It has been designed this way because there is another native PtaBs variant within the PCC 11801 genome. Nevertheless, PtaBs which come from bacillus subtilis, has higher specific activity levels than the native one. Also, it has been designed to pose a moderate strength because PtaBs is the first enzyme of the operon and so on it expresses itself higher than the others.

To achieve the integration of the expression cassette via double homologous recombination within the genome. L1-PKMCG-A is flanked by two homology regions β€œ BBa_K3726112 " 5CON1(H)_nNS5-up (PCC 11801 ) and β€œ BBa_K3726113 " 3CON5(H)_nNS5-down (PCC 11801 ). Likewise, a Spectinomycin resistance cassette is included downstream the transcriptional unit region.

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L1_PKMCG-B

...
5CON1-NSV
...
PS-PR
...
RBS PtaBs
...
PKMCG
...
Bba_0015 (5a)
...
SpecR
...
3CON5-NSV

This construct is an integrative plasmid, harboring a transcriptional unit for the expression of the half part of the MCG cycle and the enzymes that belong to the phosphoketolase pathway. If you want to know more about the MCG cycle or phosphoketolase pathway, visit the Metabolic Engineering Page.

L1-PKMCG-B, corresponds with the composite part β€œ BBa_K3726054” It is a transcriptional unit for the expression of the part β€œ BBa_K3726028” CDS_Lv0_MCG-PK which is a MoClo Lv.0 CDS element.

CDS_Lv0_MCG-PK is a polycistronic CDS that contains the half part of the enzymes of the MCG cycle and the enzymes that belong to the phosphoketolase pathway. Those enzymes are: CDS_PtaBs ( BBa_K3726019 ), CDS_PKPa ( BBa_K3726021 ), CDS_mdh ( BBa_K3726023 ), CDS_GarK ( BBa_K3726025 ), garR_CDS ( BBa_K3726027 ). 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 for cyanobacteria, PS-PR β€œ BBa_K3726084” and the strong synthetic RBS, RBS_PtaBs β€œ BBa_K3726092 ", as well as the internal RBS presented within the polycistronic CDS.

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

Alt

Since this part corresponds with the half part of a fully functional artificial carbon fixation pathway & bypass towards acetyl-CoA formation, relative expression of each enzyme required for optimal pathway functioning is controlled by their internal RBS.

As can be observed, the PtaBs initial RBS sequence have been designed to pose a moderate strength. It has been designed this way because there is another native PtaBs variant within the PCC 11801 genome. Nevertheless, PtaBs which come from bacillus subtilis, has higher specific activity levels than the native one. Also, it has been designed to pose a moderate strength because PtaBs is the first enzyme of the operon and so on it expresses itself higher than the others.

To achieve the integration of the expression cassette via double homologous recombination within the genome. L1-PKMCG-B is flanked by two homology regions β€œ BBa_K3726112 " 5CON1(H)_nNS5-up (PCC 11801 ) and β€œ BBa_K3726113 " 3CON5(H)_nNS5-down (PCC 11801 ). Likewise, a Spectinomycin resistance cassette is included downstream the transcriptional unit region.

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L1_MCGII-A

...
5CON1-NSIV
...
Prbcl-m36
...
RBS PtaBs
...
MCG-II
...
Bba_0015 (5a)
...
AmpR
...
3CON5-NSIV

This construct is an integrative plasmid, harboring a transcriptional unit for the expression of the half part of the MCG cycle. If you want to know more about the MCG cycle, visit the Metabolic Engineering Page.

L1-MCGII-A, corresponds with the composite part β€œ BBa_K3726055” It is a transcriptional unit for the expression of the part β€œ BBa_K3726038” CDS_Lv0_MCGII which is a MoClo Lv.0 CDS element.

CDS_Lv0_MCGII is a polycistronic CDS that contains the half part of the enzymes of the MCG cycle. Those enzymes are: ppc_CDS ( BBa_K3726029 ), gcl_CDS ( BBa_K3726031 ), CDS_mtkA ( BBa_K3726033 ), CDS_mtkB ( BBa_K3726035 ), CDS_mcl ( BBa_K3726037 ). Within this artificial operon 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 β€œ BBa_K3726080” and the strong synthetic RBS, RBS_Ppc β€œ BBa_K3726091 ", as well as the internal RBS presented within the polycistronic CDS.

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

Alt

SInce this part corresponds with the half part of a fully functional artificial carbon fixation pathway, relative expression of each enzyme required for optimal pathway functioning is controlled by their internal RBS.

On the one hand, Mcl and ppc enzymes are the most important enzymes of the MCG pathway. For this reason, codon optimization has been performed using DeNovo DNA Software, adjusting the codon usage for high expression in Synechococcus elongatus PCC7942. On the other hand, the rest of the enzymes that come from E.coli, though they have not been codon optimized, we designed a strong RBS to compensate for the expression levels.

To achieve the integration of the expression cassette via double homologous recombination within the genome. L1-MCGII-A is flanked by two homology regions β€œ BBa_K3726110” 5CON1(H)_nNS4-up (PCC 11801 ) and β€œ BBa_K3726111 " 3CON5(H)_nNS4-down (PCC 11801 ). Likewise, an ampicillin resistance cassette is included downstream the transcriptional unit region.

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L1_MCGII-B

...
5CON1-NSIV
...
PS-PR
...
RBS PtaBs
...
MCG-II
...
Bba_0015 (5a)
...
AmpR
...
3CON5-NSIV

This construct is an integrative plasmid, harboring a transcriptional unit for the expression of the half part of the MCG cycle. If you want to know more about the MCG cycle, visit the Metabolic Engineering Page.

L1-MCGII-B, corresponds with the composite part β€œ BBa_K3726056” It is a transcriptional unit for the expression of the part β€œ BBa_K3726038” CDS_Lv0_MCGII which is a MoClo Lv.0 CDS element.

CDS_Lv0_MCGII is a polycistronic CDS that contains the half part of the enzymes of the MCG cycle. Those enzymes are: ppc_CDS ( BBa_K3726029 ), gcl_CDS ( BBa_K3726031 ), CDS_mtkA ( BBa_K3726033 ), CDS_mtkB ( BBa_K3726035 ), CDS_mcl ( BBa_K3726037 ). Within this artificial operon CDS element is preceded by an in-silico designed RBS sequence using DeNovo DNA software.

Expression is driven by the strong constitutive promoter for cyanobacteria, PS-PR β€œ BBa_K3726084” and the strong synthetic RBS, RBS_Ppc β€œ BBa_K3726091 ", as well as the internal RBS presented within the polycistronic CDS.

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

Alt

Since this part corresponds with the half part of a fully functional artificial carbon fixation pathway, relative expression of each enzyme required for optimal pathway functioning is controlled by their internal RBS.

On the one hand, Mcl and ppc enzymes are the most important enzymes of the MCG pathway. For this reason, codon optimization has been performed using DeNovo DNA Software, adjusting the codon usage for high expression in Synechococcus elongatus PCC7942. On the other hand, the rest of the enzymes that come from E.coli, though they have not been codon optimized, we designed a strong RBS to compensate for the expression levels.

To achieve the integration of the expression cassette via double homologous recombination within the genome. L1-MCGII-B is flanked by two homology regions β€œ BBa_K3726110” 5CON1(H)_nNS4-up (PCC 11801 ) and β€œ BBa_K3726111 " 3CON5(H)_nNS4-down (PCC 11801 ). Likewise, an ampicillin resistance cassette is included downstream the transcriptional unit region.

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L1_pJAY-NSX

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5CON1-NSX
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J23119
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RBS BCD2(mod)
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sfYFP2
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Bba_0015 (5a)
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KanR
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3CON5-NSX

pJAY Constructs has been designed with a double purpose. First they are integrative plasmids for neutral sites charazterization. Expression of sfYFP2 reporter is controlled by the standard J23119 promoterBBa_K2560031 and the moderately strong RBS BCD(mod)*. Every pJAY is assembeld via Level 1 Golden Gate reaction within the AmpR RFP Dropout Lv.1 acceptor plasmid (with BBa_K2560031 Ori and with BBa_K2560031 Ori and Ampicilin Resistance BBa_K3726039.)

The sfYFP2 reporter construct is flanked by both homology arms of the required neutral site to characterize. In addition, a Kanamycin resistance marker is included for integration within the genome.

Within the original design of pJAY plasmids,the acceptor vector was planned to be a modified version of AmpR RFP Dropout Lv.1, including flanking I-SceI restriction sites. This I-SceI adapted pJAY plasmids were going to be used first for Neutral Sites characterization and after that testing the I-SceI mediated unmarking system, furtherly explained within the Engineering Page. Briefly, this plasmids would be co-integrated entirely, providing the mutants with double Kan/Amp resistance, esing its selection. Then, neutral sites relative expression could be chracterized by fluorescence. Eventually, mutants could be unmarked from the AmpR cassete (acquired from the plasmid backbone during the first single recombination event) using the I-SceI system. Since KanR cassete still within the strains, unmarked mutants could be easy selected by resistance to KanR and sensitivity to AmpR, easing the study of I-SceI system performance.

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