<img src="https://static.igem.org/mediawiki/2017/4/4d/EvaluationVectorCloning.png" alt="A scheme explaining the cloning of the Evaluation Vector.">
<img src="https://static.igem.org/mediawiki/2017/4/4d/EvaluationVectorCloning.png" alt="A scheme explaining the cloning of the Evaluation Vector.">
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<figcaption><b>Figure 2: Cloning scheme of the Evaluation Vector.</b> The Evaluation Vector was created using a specific series of cloning experiments.</figcaption></figure>Lorem ipsum dolor sit amet, consectetur adipiscing elit, sed do eiusmod tempor incididunt ut labore et dolore magna aliqua. Ut enim ad minim veniam, quis nostrud exercitation ullamco laboris nisi ut aliquip ex ea commodo consequat. Duis aute irure dolor in reprehenderit in voluptate velit esse cillum dolore eu fugiat nulla pariatur. Excepteur sint occaecat cupidatat non proident, sunt in culpa qui officia deserunt mollit anim id est laborum. Lorem ipsum dolor sit amet, consectetur adipiscing elit, sed do eiusmod tempor incididunt ut labore et dolore magna aliqua. Ut enim ad minim veniam, quis nostrud exercitation ullamco laboris nisi ut aliquip ex ea commodo consequat. Duis aute irure dolor in reprehenderit in voluptate velit esse cillum dolore eu fugiat nulla pariatur. Excepteur sint occaecat cupidatat non proident, sunt in culpa qui officia deserunt mollit anim id est laborum. Lorem ipsum dolor sit amet, consectetur adipiscing elit, sed do eiusmod tempor incididunt ut labore et dolore magna aliqua. Ut enim ad minim veniam, quis nostrud exercitation ullamco laboris nisi ut aliquip ex ea commodo consequat. Duis aute irure dolor in reprehenderit in voluptate velit esse cillum dolore eu fugiat nulla pariatur. Excepteur sint occaecat cupidatat non proident, sunt in culpa qui officia deserunt mollit anim id est laborum.
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<figcaption><b>Figure 2: Cloning scheme of the Evaluation Vector.</b> The Detailed cloning workflow which led to the finished Evaluation Vector construct with the pBS1C backbone.</figcaption></figure>We decided to additionally also provide this MCS as a BioBrick. Therefore we cloned it into the pSB1C3 backbone (via EcoRI and PstI digest) and verified the construct by sequencing. It has been submitted to the parts registry under <a target="_blank" href ="http://parts.igem.org/Part:BBa_K2273107">BBa_K2273107</a>.
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Revision as of 14:06, 24 October 2017
Short Description
Peptidosomes in combination with Bacillus subtilis offer a perfect platform for enhanced protein overproduction by the means of efficient protein secretion and facilitated purification due to the peptidosomes’ pores. Naturally, B. subtilis is a strong secretion host and in order to take full advantage of this great potential it is necessary to evaluate all possible combinations of the B. subtilis’ secretion signal peptides and the proteins of interest. Therefore, we developed the Evaluation Vector (EV) which is an efficient genetic tool with a specially designed multiple cloning site (MCS) to easily exchange translational fusions of the desired protein with the secretion signal peptides.
Background
Tool development and their proper evaluation are core aspects of Synthetic Biology. In our project EncaBcillus one main idea was to establish Peptidosomes with encapsulated Bacteria as efficient protein overproduction platform. We took advantage of B. subtilis’ ability to efficiently secrete proteins into its environment in order to increase overall yields and to simplify the purification of the desired proteins. Therefore, we developed a general expression evaluation vector (EV) with easily exchangeable units: I) allowing the replacement of the promoter (which drives the system) and II) a multiple cloning site enabling to work with translationally fused composite parts. In our case, a typical composite part consists of a signal peptide (for secretion in B. subtilis) and a protein of interest.
In summary, our EV was designed to fulfill the following distinct features:
Exchangeable promoter region
Insertion of transcriptional and translational expression units
Fulfilling the RFC10 and RFC25 BioBrick standard
Easy cloning and screening procedure in Escherichia coli
As our project is based on the Gram-positive model organism B. subtilis, we decided to use a previously well-evaluated B. subtilis vector as source for our Evaluation Vector: the integrative vector pBS1C1. In brief, the vector has the following features for cloning in E.coli: an ori of replication and the bla gene mediating resistance against ampicillin. The B. subtilis specific part of the vector contains the multiple cloning site (MCS), a cat cassette providing resistance against chloramphenicol and flanking regions needed for integration into the amyE locus. After integration into α-amylase. The resulting disruption of the native gene leads to a loss of this enzymatic activity, thereby making it a vector easy to screen for by performing a starch test for positive integration events. (For a detailed description of the original vector features please have a look at Radeck et al., 2013 and our Design section of the EV.)
1
J Radeck, K Kraft, J Bartels, T Cikovic, F Dürr, J Emenegger, S Kelterborn, C Sauer, G Fritz, S Gebhard and T Mascher "The Bacillus BioBrick Box: generation and evaluation of essential genetic building blocks for standardized work with Bacillus subtilis" Journal of Biological Engineering 7:29 (2013). PubMed
Design
As described above, we chose to modify the backbone of pBS1C11 to create our new Evaluation Vector (EV), by engineering the multiple cloning site (MCS) according to the scheme below.
Finally, we combined our new MCS, by ligating the digested RFPsyn2 (cut with XbaI and AgeI) with the lacZα fragment (cut with AgeI and PstI). This MCS was inserted into the pBS1C-PxylA backbone, which was prior opened using BsaI (resulting in an XbaI overhang) and PstI (Figure 2). The final construct of our EV was verified by sequencing.
Figure 2: Cloning scheme of the Evaluation Vector. The Detailed cloning workflow which led to the finished Evaluation Vector construct with the pBS1C backbone.We decided to additionally also provide this MCS as a BioBrick. Therefore we cloned it into the pSB1C3 backbone (via EcoRI and PstI digest) and verified the construct by sequencing. It has been submitted to the parts registry under BBa_K2273107.
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