CRISPR/Cas systems are powerful tools that have gained a lot of popularity in the recent years. As they can be used for a wide array of applications - like the integration of whole genes, alteration of single nucleotides, knock-outs of whole genetic regions, as well as the use of the DNA-binding property in a multitude of applications through so called deadCas systems, where the Cas protein does not exhibit nuclease activity (Hsuet al., 2014) - we were eager to implement such a system into our own toolbox. Diving into the literature we noticed many different systems are available,the most commonly used one being CRISPR/Cas9, and we began to wonder which of them we should use.
In our description we presented CRISPR/Cas9 and CRISPR/Cas12a, showing the differences of these two systems. Looking deeper into CRISPR/Cas12a we noticed a few advantages that finally led us to choose it as our preferred system.
As the sgRNA used as a guide for Cas9 is usually ~100nt long, chemical synthesis is more complex and expensive in comparison to the ~43nt needed for the Cas12a guiding crRNA (Swarts and Jinek, 2018) - an unpleasant fact, especially for iGEM teams that do not have many resources available to them, but the main reasons we chose Cas12a are others. Multiplexed gene editing is one of the key features of these CRISPR/Cas systems, but how to actually apply it differs:
For Cas9 each sgRNA is in need of its own promoter, which means that they have to be expressed from different vectors or a multi cassette vector ( X. Ma et al., 2015; Z. Zhang et al., 2016 ). In contrary, multiplexed genome editing with Cas12a can be achieved simply by expressing all of the needed guide RNAs in one transcriptional unit, where they are then processed into different crRNAs by Cas12a (Kim, et al., 2016; Nishimasu et al., 2017). This is a huge advantage of Cas12a. Furthermore, CRISPR/Cas9 was shown to be toxic in cyanobacteria (Wendt et al., 2016), which is one of the foremost reasons CRISPR technologies have not been widely applied in cyanobacteria - the usage of Cas12a though, does not seem to have the same toxicity Ungerer and Pakrasi, 2016 , making it the ideal candidate for the Green Extension of the Marburg Collection.
The actual implementation of the CRISPR/Cas12a system into our toolbox necessitated a well thought out plan. The design of our CRISPR/Cas12a system was mainly affected by the fact that we wanted to have a convenient and rapid tool for genomic manipulation. The lvl 0 part (What are parts? Read more about it click here!) of the Cas12a protein was created via PCR amplification from the plasmid pSL2680, but special overhangs were added in order to clone the PCR product into a lvl 0 acceptor vector. The part was introduced as a coding sequence (CDS) part in the MoClo standard to be included in the Green Expansion of the Marburg Collection. The lvl 1 part of the Cas12a protein was equipped with a rather weak promoter so that the toxicity caused by overproduction of the endonuclease could be kept low. The parts used for lvl 1 assembly were: pMC0_1_03 + pMC0_2_03 + pMC0_3_07 + pMC0_4_33 + pMC0_5_07 + pMC0_6_17. For the construction of the crRNA part the design of the plasmid pSL2680 was mainly maintained, but the lacZ cassette was replaced by a GFP cassette to enable easier screening of crRNA assembly and to reduce expenses for X-Gal/IPTG. It was constructed as a part reaching from the RBS site to the end of the terminator site. As the whole system is built for modular cloning in PhytoBrick syntax, it is possible to freely exchange the parts around the Cas12a and crRNA parts - in this way the amount of crRNA/Cas12a can be controlled by choosing promoters with different strengths.
Our initial plan was to synthesize the crRNA with the desired overhangs, but as the sequence contains multiple direct repeats, it was not possible for providers to synthesize this construct, which is why we split it into four different parts that then had to be assembled. For this assembly the four parts were first cloned into the pJET1.2/blunt vector by Thermo Scientific and then digested with BsaI while the acceptor vector was digested with BsmBI. In this way the final vector still contains BsaI recognition sites, so that it can be used in a level 1 assembly Golden Gate reaction. The cloning of the level 2 part with this crRNA part was done by ending with a ligation step to make sure the GFP dropout remains in the vector.

The Marburg Collection is a toolbox from last year’s iGEM Marburg team for the rational design of metabolic pathways and genetic circuits or any other DNA construct. Thanks to its flexible design based on the ‘Dueber toolbox’ design from Lee et. al (2015) it can be used in a multitude of chassis: since it complies with the PhytoBrick standard, it can even be extended to eukaryotic chassis such as plants. The design of the toolbox is rather simple and user friendly: LVL 0 parts are the basic foundation of every assembly. They contain a single genetic element such as a promoter or terminator. Up to 8 LVL 0 parts are used to build a LVL1 plasmid containing a single transcription unit. Up to 5 of these transcription units can be assembled together in a LVL 2 plasmid (Lee et. al (2015)).

Design of placeholders

Here we present a new feature of the Marburg Collection 2.0: Placeholders. These parts make it possible to construct plasmids with a placeholder, which can be later on exchanged with any part of the same type.
A key feature in our expansion is the addition of placeholders that allow high throughput assembly of plasmids that only differ in one part. A promoter placeholder for example is built into a LVL 1 construct at the promoter position. Instead of a promoter however it contains a GFP cassette and reversed BsaI cutting sites. This allows BsaI cleavage and removal of the GFP cassette even after assembly, due to the fact that the BsaI recognition site is not removed from the placeholder.

After that any promoter of choice can be inserted at that position. After ligation, no BsaI cutting sites remain on the vector, so in the end mainly the newly assembled remains. These steps also happen in a one pot one step reaction just like any other Golden Gate assembly.

White green selection under UV light can be used to determine the colonies with the right plasmid: green ones still contain the plasmid with a placeholder, white ones contain the desired vector.

Available Placeholders

Placeholders exist for every position from 1-6, but technically placeholders can also span multiple positions to insert multiple parts at once. For example a placeholder for the position promoter and RBS could be replaced with any combination of promoter and RBS that is deemed right for a specific application. This however would then be a three part assembly. The application of such parts is so narrow that we decided to build the most useful ones. Thanks to its clever design the construction of more placeholder is so simple that it can be done by the user himself with a single site directed mutagenesis of a flank.

Design of the first panS based MoClo compatible shuttle vector

The heart piece of green expansion is BBa_3228069, a LVL 0 part containing origins of replication for E. coli and S. elongatus as well as a spectinomycin cassette. It resembles a type 7+8 (antibiotic cassette + ori) composite part and can be seen as the cyanobacteria specific LVL1 entry vector. Another version of this entry vector contains a kanamycin cassette and BsmbI cutting sites and can be used as the LVL2 entry vector. Just like in our LVL 0 entry vectors for basic parts, we prompted for a fluorescence based reporter in the dropout, rather than lacZ for blue/white screening. Therefore both vectors contain an RFP dropout to signal an insertion. Using this vector in our updated Golden Gate assembly protocols, we achieve a rate of about 9:1 white to red colonies, showing that the assembly is rather efficient.

Designing the characterization of BioBricks

Here we present the design of the plasmids and the workflow used to characterize BioBricks.
In order to characterize BioBricks they need to be inserted into a measurement vector that is stably maintained in cyanobacteria. The design of the plasmids to characterize our parts was an amazing experience as it was one of the first times that we acted not only as creators but also as users of our toolbox. Therefore design of the workflow and design of new parts was tied together very closely.
The criteria that the measurement vectors need to meet are some of the most basic principles of Synthetic Biology:
In order to be comparable, all of the constructs must be almost identical and only differ in the part to be tested. Instead of building each construct independently we utilized our placeholders (See Results: placeholder) to build all measurement plasmids for the same type of part from the same blueprint.
We present a set of measurement entry vectors for the characterization of BioBricks in cyanobacteria (Part range BBa_K3228073 to BBa_K3228075 as well as BBa_K3228090). They contain our MoClo compatible shuttle vector for cyanobacteria BBa_K3228069 and are therefore the only MoClo based vector for the characterization of BioBricks in cyanobacteria. These pre assembled LVL 1 plasmids contain a placeholder for their respective BioBrick type that acts as a Dropout to quickly and effortlessly insert any part of the same type for an easy characterization. In our results we show how these measurement entry vectors can save a lot of effort and money when characterizing a greater library of parts. Additionally, the usage of the same entry vector for each measurement will aid in greater comparability and reproducibility.
For greater comparability across other data sets we decided to use similar BioBricks as in measuring the toolbox for Vibrio natriegens in the last year. The design from there on was pretty straight forward for promoter and RBS.

For terminators however the design is a bit more intricate: a terminator is not measured in its activity but rather in its isolative power. Hence, a strong terminator should result in a weak signal. On top of that, measuring the activity both upstream and downstream of the terminator with two independent reporters would give insight on the exact transcriptional activity around the area of the terminator (Chen et al., 2013), resulting in the most accurate results in respect to the molecular dynamics of a terminator (See: modeling).
A LVL 2 plasmid was logically the easiest way to construct such a part. We designed a normal LVL 1 plasmid containing an mTurqouise reporter and a secondary LVL 1 plasmid containing an YFP reporter but missing a promoter.

The fraction of the signal strength of YFP and mTurquoise describe the isolative capacity of the terminator best (Chen et al., 2013).
This way of calculating isolative strength is also used in RNA-seq to determine the strength of terminators.

Modular Engineering of Genome Areas (M.E.G.A.)

Here we represent an expansion to the Marburg Collection 2.0: M.E.G.A. – a set of parts for the genomic integration of genes in Synechococcus elongatus UTEX2973 and other cyanobacteria that can be easily extended to other chassis. This set includes parts with homologous flanks for homologous recombination as well as a necessary set of new terminators and antibiotic resistances.

Finding new artificial Neutral integration Site options (a.N.S.o.)

Artificial neutral integration Site options (aNSo) for our purpose in Synechococcus elongatus needed to fulfil three criteria, to be genuinely considered as potential candidates.
A highly precise algorithm was implemented in a Python script to find these potential candidates (link to modeling) by describing the following criteria. First, no gene and transcription start site (TSS), i.e. no CDS, was allowed to be disturbed, assuring that no lethal modification was created by integration. Thereby, we searched for intergenic regions where no TSS had been identified, with a length of at least 500 bp. These sequences had to be extended in both 3’ and 5’ direction up to a length of at least 2500 bp providing flanks to ensure the integration by homologous recombination, which should be performed in the lab subsequently. In the middle of these sequences any gene of interest can be inserted, which gets integrated into the genome by the mentioned homologous recombination, due to homologous flanks. Second, integration site sequences were not allowed to contain restriction sites that interfere with the iGEM standards to simplify the cloning process and make them more cross compatible. All sequences that contained such restriction site were discarded. Executing this newly developed and unique algorithm resulted in two unique aNSo's within the genome of S. elongatus.

Design neutral integration sites

For a successful homologous integration the sequence to be integrated needs to be flanked by two integration sites homologous to the neutral site on the target genome. Additionally, the integrated sequence needs to contain an appropriate selection marker to be able to select for integration events.
It is included in the syntax of the Marburg Collection, that the positions 1 and 6 can not only be used for connectors but for integration sites as well. Since integration sites contain a BsmBI restriction site just like a connector part, their construction is a bit more intricate than a normal part:

In a LVL 1 construct, the positions 2-5 representing a full transcription unit (promoter, RBS, CDS, terminator) would be integrated into the genome, while positions 7-8 (origin of replication,antibiotic cassette) would be cut off in the recombination event. The issue with this assembly would be that a marker for the selection after integration is completely missing. Hence, we decided to split the position of the terminator in a similar fashion in which C-terminal tags were integrated into the syntax last year:

All terminators of the Marburg Collection were rebuild as "5a" parts similar to C-terminal tags. This allowed to insert an antibiotic cassette at the position "5b". For this position 4 different antibiotic cassettes were designed.
Our integration sites were also designed as connectors, so it is possible to build a gene cascade with up to 5 genes that can be inserted into a single neutral site. All integration sites function as 5'Con1 and 3'Con5 connectors, meaning they are always at the beginning of the first and the end of the last gene in a LVL2 construct.
It is important to note for the user that when designing the vector for integration, the origin should not be compatible with the organism. This way, it enters the organism and then integrates into the genome or disappears as it cannot be replicated in its new host. Otherwise the vector will be maintained in the transformed organism and it will be rather complicated to remove it. If there is no compatible origin available. We designed our toolbox so that it can always be digested with NotI to linearize the integration cassette and extracted it over a gel. In a lot of cases transformations and homologous recombinations with linear DNA are a lot more efficient. (See results of strain engineering)
Our system offers the integration of up to 5 genes with 4 different selection markers at 5 different integration sites. Therefore, the integration of up to 20 genes into the UTEX wild type genome is possible.

Fluorescence reporters for characterization of parts

When working in Synthetic Biology, reporter genes such as fluorescence proteins are indispensable elements to characterize BioBricks. For a good characterization a suitable reporter is required. But reporters can be more than just merely a detection tool for transcriptional activity but they can also give a deeper insight into cellular conditions beyond the genetic context. We provide a diverse set of reporters not only for the purpose of describing genetic tools but also for the sensing of a variety of parameters which are crucial for cyanobacteria.

eYFP

Aequorea victoria
Excitation Maximum (nm)	515
Emission Maximum (nm)	527
Extinction Coefficient (M-1 cm-1)	67,000
Quantum Yield	0.67
Brightness	44.89
pKa	6.9
Maturation (min)	9.0
Life- span (ns)	3.1

Source: FP Base (EYFP)

eYFP is the mutant of green fluorescent protein naturally occuring in Aequorea victoria. It is a preferred reporter for cyanobacteria as it bypasses the wavelength at which absorption photoactive pigments occurs, resulting in stronger signal overall (Kukolka & M. Niemeyer, 2004).

Additionally, autofluorescence of cyanobacterial cells is rather low at that point, resulting in a stronger signal compared to the background, increasing the resolution of characterizations.

sYFP2 (S.e.)

Aequorea victoria
Excitation Maximum (nm)	515
Emission Maximum (nm)	527
Extinction Coefficient (M-1 cm-1)	101,000
Quantum Yield	0.68
Brightness	68.68
pKa	6.0
Maturation (min)	4.1
Life- span (ns)	2.9

Source: FP Base (sYFP2)

sYFP is a superfolded version of YFP. Thanks to faster maturation it leads not only to a twofold signal strength compared to eYFP: the fast maturation also ensures that every transcribed mRNA leads to the same amount of correctly folded fluorescent protein. This makes measurements more robust towards varying cellular contexts.

mTurquoise2 (S.e.)

Aequorea victoria
Excitation Maximum (nm)	434
Emission Maximum (nm)	474
Extinction Coefficient (M-1 cm-1)	30,000
Quantum Yield	0.84
Brightness	25.2
pKa	4.5
Maturation (min)	112.2
Life- span (ns)	3.7

Source: FP Base (mTurquoise2)

mTurquoise2 is a brighter fluorescent variant of mTurquoise with faster maturation and a high photostability, making it one of the better for microscopy applications. Thanks to a shifted emission maximum it is possible to detect both, YFP and mTurquoise in single cells with virtually no bleed-through of signal, making it suitable for dual fluorescent protein applications like terminator characterization ( Link to johannas characterization text).

NanoLuc

NanoLuc is a small luminescent reporter with just a molecular weight of 19,5 kDA. This reporter stands out with a signal strength that is orders of magnitude higher than compared traditional luminescent reporters. It is a very small protein and unlike the lux operon it is only a single gene, reducing the metabolic burden onto the host to a bare minimum. Additionally it is not using ATP as a substrate which is a valuable energy resource in cells. This way it does not affect the cellular context and acts as a truly orthogonal reporter.

TeLuc

TeLuc is a triple mutant of NanoLuc. Thanks to a modified substrate binding pocket it is able to use DTZ as a substrate, resulting in a (42 nm) red-shift (from 460 nm to 502 nm peak) of emission. This bypasses the absorption of Chlorophyll A, making it the more suitable reporter for phototrophic organism.

Antares2

Antares2 is a coupled bioluminescence protein consisting of TeLuc and two flanking CyOFP fluorescence reporters. It abuses the Bioluminescence Resonance Energy Transfer (BRET) to excite CyOFP with the luminescence of TeLuc. This results in a further red-shift, making it suitable for applications like deep tissue analysis. Additionally, it can be used in conjunction with NanoLuc thanks to the utilization of two distinct substrates as well as varying emission peaks. Therefore it is the world’s only dual luminescent detector pair.
Luminescence is a great tool for accurate measurements, but in the world of biosensors for the detection of cellular conditions only fluorescent reporters are established yet. We present reporters for the two most important chemical parameters in cyanobacteria: pH and redox status. We saw that the pH of the media has a significant impact on the growth of the culture (Link to results growth rate), which is previously described (Kallas, Castenholz et al.). Cyanobacteria are not equipped to regulate their internal pH very well, yet they still depend on a stable proton gradient to keep up their photosynthetic machinery (Billini et al.). We present phlurion2, a reporter that is modulated in its excitation peak by varying ph values.

pHlurion2 (S.e.)

Info Box:

Aequorea victoria	acidic (pH 5,5)	alkaline (pH 7,5)
Excitation Maximum (nm)	395	475
Emission Maximum (nm)	509	509

Source: FP Base (pHlurion2)

pHlurion2 is a mutant of GFP2. Its excitation maximum depends on the surrounding pH value. Therefore it can be used to detect changes in the cellular pH. As described above a biosensor for this parameter could be of great use, especially in cyanobacteria. (Mahon, 2011)
Another important cellular factor is the internal redox status. During photosynthesis reactive oxygen species (ROS) are constantly produced as a byproduct. A critical mass of reactive oxygen species leads to serious cell damage and cell toxicity through chemical alterations of proteins, DNA and lipids. Especially under high light conditions the redox status becomes a crucial parameter as it can threaten the cellular fitness.
For example, the overexpression of orthogonal thioredoxin peroxidase leads to the degradation of ROS resulting in enhanced growth of PCC7942, (Kim et al.) We present rxYFP, a redox-sensitive reporter for cyanobacteria.

rxYFP (S.e.)

Aequorea victoria
Excitation Maximum (nm)	515
Emission Maximum (nm)	527
Extinction Coefficient (M-1 cm-1)	101,000
Quantum Yield	0.68
Brightness	68.68
pKa	6.0
Maturation (min)	4.1
Life- span (ns)	2.9

Source: FP Base (sYFP)

rxYFP is a redox-sensitive yellow fluorescent protein deriving from Aequorea victoria GFP. This reporter contains a pair of redox-active Cys residues (Cys149 and Cys202), which are connected through a disulphide bond under oxidative conditions, resulting in a 2.2-fold reduction of the emission peak. This allows to determine the redox potential in the environment which then expressed the output of fluorescence.