Part:BBa_K3781105

weird_plex, MocloMania expression vector

This basic part codes for weird_plex, the heart and soul of our MocloMania collection. It is a plasmid that acts both as a L1 destination vector for Modular Cloning assembly of basic parts and as an expression vector for recombinant protein expression in Leishmania tarentolae. In order to meet both of these demands, weird_plex is equipped with a very special set of features.

Its sequence is based on the commercially available Leishmania expression vector pLEXSY_I-blecherry3 distributed by german biotech company Jena Bioscience in their LEXSinduce3 Expression Kit. Jena Bioscience has specialized on selling Leishmania-adapted expression kits that facilitate recombinant gene expression in the protozoan expression host.

The pLEXSY_I-blecherry3 plasmid is an integrative expression vector, meaning that the introduced target gene sequences are integrated into the Leishmania genome after cell transfection. Genes of interest are introduced into the vector with the help of two multiple cloning sites. This insertion region is controlled by a T7 promoter, a DNA sequence specifically bound by the constitutively expressed T7 RNA polymerase. T7 polymerase is an E. Coli phage derived polymerase that, together with its promoter, mediates strong gene expression. Another cool feature of the pLEXSY_I-blecherry3 plasmid is its inducible gene expression. This is achieved by a downstream tet operator which is part of a tetracycline-controlled activation system commonly found in eukaryotic expression hosts. Together with the constitutively expressed tet repressor, this operon suppresses binding of the T7 polymerase to its promoter, but can easily be de-activated by the addition of tetracycline to the culture medium.

Furthermore, the pLEXSY_I-blecherry3 plasmid contains a bleomycin resistance that, after addition of bleomycin to the culture medium, acts as an important selection marker for screening of successfully transfected cells. The resistance coding gene is directly fused to an mCherry coding gene. Since both these genes are located downstream of the insertion region and thus under control of the T7 promoter, the production of mCherry correlates with the production of target protein. Monitoring mCherry fluorescence can thus be used to determine successful induction and productive target gene expression.

Beyond this, the plasmid's cloning frame also includes a Leishmania mexicana derived secretion signal peptide as well as a C-terminal Hexa-histidine-tag, flanked on either end by multiple restriction sites. Thus, pLEXSY_I-blecherry3 allows for either cytosolic or secretory protein expression and optional His-tagging of the target protein. During domestication of the plasmid towards the MoClo system, these two features were eliminated. Instead, their sequences were adapted and turned into functional L0 basic parts within the MocloMania collection, the sAP secretion tag and the Strep8His-tag. This way, switching between cytosolic and secretory protein expression is easily coordinated within a simple MoClo reaction.

Additional to the genetic optimizations towards expression in Leishmania, pLEXSY_I-blecherry3 also contains an E. Coli expression cassette that consists of an ampicillin resistance gene as well as a high copy number E. Coli origin of replication. These allow for the insert-carrying vector to be transformed and amplified in E. Coli which is important because transfection success relies on the input of sufficient DNA amounts. This expression cassette is designed to be cleaved off by the enzyme SwaI prior to transfection, leaving behind a linear DNA doublestrand containing all the genes relevant for transgenic protein expression in Leishmania.

In order to make the pLEXSY_I-blecherry3 vector suitable for our Modular Cloning approach, it first had to be domesticated towards our cloning system. This was done by eliminating three endogenous BsaI restriction sites via the introduction of single point mutations. Furthermore, the multiple cloning sites were used to introduce a LacZ-alpha gene cassette into the vector backbone. When transformed with a LacZ-omega carrying E. Coli strain and grown on IPTG/XGAL agar plates, this will result in blue appearing colonies. .

This doesn't only make the vector accessible to blue-white screening, it also allowed for new BsaI restriction sites to be introduced into the plasmid, flanking either end of the lacZ-alpha gene in inverted orientation. These restriction sites take on the function of the multiple cloning sites during classical cloning by enabling the insertion of L1 MoClo constructs into the vector.

With the help of all these domestication steps we were able to turn pLEXSY_I-blecherry3 into weird_plex, a jack of two trades. Any basic part within our MocloMania collection can be assembled into a cohesive L1 construct and introduced into weird_plex in one single MoClo reaction.

level 1

size 7710 bp

antibiotic resistance ampicillin, bleomycin

cloning positions B2-B5

Data

As the plasmid vector used for transfecting and expressing target gene constructs into Leishmania tarentolae, weird_plex is of outstanding importance to our MocloMania collection. All of its features mentioned in the introduction text are visualized in the schematic plasmid map in Figure 1, displaying both the original pLEXSY_I-blecherry3 plasmid as well as our domesticated version, weird_plex.

• 

  Figure 1 | Plasmid maps for both pLEXSY_I-blecherry3 and weird_plex
  orange markings | pLEXSY_I-blecherry3 | pre-existing endogenous BsaI restriction sites
  orange markings | weird_plex BsaI restriction sites introduced for MoClo adaptation
  

Due to the significance of this L1 expression vector to the implementation of our project, its domestication was the first thing that we started to work on. The experimental plan of eliminating the endogenous BsaI restriction sites by introducing silent point mutations via PCR seemed very straight-forward, yet proved to be far more difficult than initially expected. In numerous attempts of PCR amplification, one of the fragments repeatedly turned out to be flawed, hindering successful ligation of the final vector. After several unsuccessful attempts at amplifying the desired three PCR fragments, we decided to divide the problematic fragment into several smaller parts. Further attempts with new primers seemed to reveal the underlying problem regarding the amplification of the DNA fragment. After identifying a problematic 400 bp sequence between the primers plex-400-rev and plex-2-for, we decided on trying to amplify the sequence again which lead to promising PCR results, seen in Figure 2.

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  Figure 2 | PCR fragments for pLEXSY_I-blecherr<3 domestication
  1 | plex_3-forward + plex_1-reverse | 1886 bp
  2 | plex_1-forward + plex_2-reverse | 1718 bp
  3 | plex_2-forward + plex_400-reverse | 475 bp
  4 | plex_2-forward-verlängert + plex_400-reverse | 480 bp
  5 | plex_400-forward + plex_J-reverse | 1877 bp
  6 | plex_400-forward + plex_N-reverse | 1623 bp
  7 | plex_J-forward + plex_3-reverse | 2221 bp
  8 | plex_N-forward + plex_3-reverse | 2638 bp
  M | ThermoFisher Scientific GeneRuler DNA Ladder Mix [bp]

After gel extraction and ligation of the PCR fragments, the resulting plasmid was analysed for its genetic sequence via commercial Sanger sequencing. This revealed that there was an estimated 150 bp fragment missing from our assembled plasmid. This 150 bp fragment was identified to be a highly repetitive region within pLEXSY_I-blecherry3, see labelled FU, consisting of at least twelve consecutive C bases. This lead us to assume that Q5 DNA polymerase might struggle with replicating such a highly repetitive base pair sequence.

• 

  Figure 3 | Sequencing results for two versions of weird_plex
  A | first domestication attempt | 150 bp missing
  B | final domestication attempt | no bp missing
  

In Figure 3, A shows the sequencing results of our first attempt at domestication that revealed the missing 150 bp region suspected to cause problems with PCR amplification. B shows the sequencing result of our final cloning product weird_plex, after successful domestication.

With the help of several different DNA templates, one of which was another commercially available expression vector by Jena Bioscience GmbH named pLEXSY_IE-blecherry4, we tried to amplify the problematic sequence once again. This time, it was supposed to be part of a 480 bp PCR fragment, which had to be amplified out of the plasmid and thankfully didn't contain any BsaI restriction site.

• 

  Figure 4 | 480 bp PCR fragment containing problematic region amplified from different plasmid templates
  1 | pLEXSY_I-blecherry3
  2 | weird_plex | first domestication attempt
  3 | pLEXSY_IE-blecherry4
  L | Thermofischer GeneRuler Plus Ladder [bp]

Figure 4 shows the resulting PCR fragments amplified from the different plasmid templates. It can be seen, that the previous version of our domesticated vector, lane 2, is missing basepairs which are present in the original undomesticated expressions vectors pLEXSY_I-blecherry and pLEXSY_IE-blecherry4. Yet the fragment in lane 3 runs a bit higher than the one in lane 1, which lead to us using this amplificate, originating from pLEXSY_IE-blecherry4, for further ligation attempts. The attempt did improve the quality of the PCR amplificate but it did not completely solve the problem, as sequencing revealed that there were still estimately 30 bp missing from the plasmid. Thus, a new strategy was employed, relying on conventional insertion cloning of the problematic region out of the original pLEXSY_I-blecherry3 into our otherwise domesticated vector. In order not to re-introduce any of the endogenous BsaI recognition sites, only a specific set of restriction enzymes could be used for this, one of which was ClaI, which is blocked by Dam-methylation. In order to bypass this issue, the relevant DNA fragments were transformed into CGSC5127, a Dam- E. coli strain, followed by plasmid miniprep and digest with ClaI and SpeI.

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  Figure 5 | Restriction digest using ClaI and SpeI
  1 | pLEXSY_I-blecherry3 | undigested | 8210 bp
  2 | pLEXSY_I-blecherry3 | ClaI | 8210 bp
  3 | pLEXSY_I-blecherry3 | SpeI | 8210 bp
  4 | pLEXSY_I-blecherry3 | ClaI + SpeI | 6240 + 1970 bp
  5 | weird_plex | first domestication attempt | undigested | 7710 bp
  6 | weird_plex | first domestication attempt | ClaI | 7710 bp
  7 | weird_plex | first domestication attempt | SpeI | 7710 bp
  8 | weird_plex | first domestication attempt | ClaI + SpeI | 5740 + 1970 bp
  9 | weird_plex | first domestication attempt | ClaI + SpeI | 5740 + 1970 bp
  L | Thermofischer GeneRuler Plus Ladder [bp]

Figure 5 shows the result of the restriction digest with ClaI and SpeI. Lane 4 and 8 show the results of the double digest as expected in silico. The respective lanes where thus extracted from the gel for further cloning. Ensuing ligation of the troublesome fragment of pLEXSY_I-blecherry3 into the domesticated weird_plex backbone brought out a correctly assembled plasmid, which could be proven to be the successfully domesticated and intact weird_plex vector. This could be shown both by restriction digest, see Figure 7, as well as sequencing, see Figure 3.

Other than the elimination of endogenous BsaI restriction sites, another important step in domesticating our expression vector for Modular Cloning was the introduction of the lacZ-alpha gene cassette into the plasmid. This allows for blue-white screening after cloning and transformation into LacZ-omega carrying E. coli cells. Along with the lacZ-α gene cassette, new BsaI restriction sites were introduced into the plasmid in order to function as a sort of multiple cloning site for insertion of MoClo L1 constructs into our expression vector using BsaI.

Figure 6 shows the successful amplification of the lacZ-alpha gene cassette originating from pICH47742, a plasmid backbone acquired from the Chlamy MoCLo Toolkit established by Crozet et al. After amplification, the lacZ-fragment as well as the domesticated destination vector were sequentially digested with restriction enzymes BglII and NotI and ligated using T4 DNA ligase, leading to the finished and ready-to-use expression vector weird_plex.

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  Figure 6 | PCR of lacZ-alpha fragment
  1 | lacZ-alpha | 623 bp
  L | Thermofischer GeneRuler Plus Ladder [bp]
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  Figure 7 | Test digest of L1 expression vector using SacI
  1 | pLEXSY_I-blecherry3 | 3968 + 2515 + 1727 bp
  2 | weird_plex | 3927 + 2515 + 1268 bp
  L | Thermofischer GeneRuler Plus Ladder [bp]

Figure 7 shows a test digest of the original vector pLEXSY_I-blecherry3 compared to the domesticated expression vector weird_plex. Along with Sanger sequencing of the whole plasmid sequence this provided definite proof of the successful domestication and assembly of the weird_plex expression vector.

The MocloMania collection

This plasmid vector is the core to the MocloMania collection, the very first collection of genetic parts specifically designed and optimized for Modular Cloning assembly and recombinant protein expression in the protozoan parasite Leishmania tarentolae.

Are you trying to express complexly glycosylated proteins? Large antibody side chains? Human proteins that require accurate post-translational modification? Then Leishmania might be just the right organism for you! Leishmania tarentolae’s glycosylation patterns resemble those of human cells more closely than any other microbial expression host, while still delivering all the benefits of microbial production systems like easy transfection and cultivation. So instead of relying on mammalian cell lines, try considering Leishmania as your new expression host of choice!

Our MocloMania collection will allow you to easily modify your protein of choice and make it suitable for downstream detection and purification procedures - all thanks to the help of Modular Cloning. This cloning system was first established by Weber et al. in 2011 and relies on the ability of type IIS restriction enzymes to cut DNA outside of their recognition sequence, hereby generating four nucleotide overhangs. Every basic part in our collection is equipped with a specified set of overhangs that assign it to its designated position within the reading frame. These so-called cloning positions are labelled B2-B5 from upstream to downstream. By filling all positions with the basic parts of your choice, you can easily generate variable genetic constructs that code for the fusion protein of your desire.

We furthermore provide a specifically domesticated Leishmania expression vector, named weird_plex, which will package your fusion construct into a functional transcriptional unit that is optimized for high expression in Leishmania.

The best part? Because of the type IIS restriction properties and the specifity of the generated overhangs, restriction and ligation of your construct can all happen simultaneously in a simple one-step, one-pot reaction. This will safe you a lot of time and frustration in your cloning endeavours!

Do we have your attention? In the table below you can find some basic information on how our cloning system, along with most other MoClo systems, is set up. Please feel free to check out our wiki to find more information on Leishmania and Modular Cloning as well as to understand how the part that you are looking at integrates into our part collection. See you there!

MOCLO | Important nomenclature and parameters

Level	What does this level contain?	antibiotic resistance	Enzyme used for ligation
L0	The foundation to every MoClo construct which are basic genetic units, such as coding sequences, promoters, terminators	spectinomycin	BbsI
L1	Several L0 parts assembled into a functional transcriptional unit, e.g. consisting of promoter, coding region and terminator	ampicillin	BsaI
L2	Multiple transcriptional units added into one multi-gene construct, e.g. a protein of interest fused to a resistance cassette	kanamycin	BsaI

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal EcoRI site found at 1689
  Illegal EcoRI site found at 2487
  Illegal XbaI site found at 2514
  Illegal SpeI site found at 5191
  Illegal PstI site found at 2526
  Illegal PstI site found at 3508
  Illegal PstI site found at 4831
  Illegal PstI site found at 5822
  Illegal PstI site found at 6579
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal EcoRI site found at 1689
  Illegal EcoRI site found at 2487
  Illegal NheI site found at 1008
  Illegal SpeI site found at 5191
  Illegal PstI site found at 2526
  Illegal PstI site found at 3508
  Illegal PstI site found at 4831
  Illegal PstI site found at 5822
  Illegal PstI site found at 6579
  Illegal NotI site found at 2737
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal EcoRI site found at 1689
  Illegal EcoRI site found at 2487
  Illegal BglII site found at 2130
  Illegal BamHI site found at 2508
  Illegal BamHI site found at 4085
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal EcoRI site found at 1689
  Illegal EcoRI site found at 2487
  Illegal XbaI site found at 2514
  Illegal SpeI site found at 5191
  Illegal PstI site found at 2526
  Illegal PstI site found at 3508
  Illegal PstI site found at 4831
  Illegal PstI site found at 5822
  Illegal PstI site found at 6579
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal EcoRI site found at 1689
  Illegal EcoRI site found at 2487
  Illegal XbaI site found at 2514
  Illegal SpeI site found at 5191
  Illegal PstI site found at 2526
  Illegal PstI site found at 3508
  Illegal PstI site found at 4831
  Illegal PstI site found at 5822
  Illegal PstI site found at 6579
  Illegal NgoMIV site found at 1675
  Illegal NgoMIV site found at 4368
  Illegal NgoMIV site found at 4429
  Illegal NgoMIV site found at 5364
  Illegal NgoMIV site found at 5598
  Illegal NgoMIV site found at 5669
  Illegal NgoMIV site found at 6734
  Illegal AgeI site found at 5412
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI site found at 2726
  Illegal BsaI.rc site found at 2142
  Illegal SapI site found at 2090