Difference between revisions of "Team:TU Dresden/Composite Part"

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<img src="https://static.igem.org/mediawiki/2017/7/7e/EvaluationVector.png" alt="A scheme explaining the design of the Evaluation Vector.">
 
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<figcaption><b>Figure 1: New layout of the multiple cloning site in our Evaluation Vector.</b> The crosses indicate restriction enzyme sites: E= EcoRI, N= NotI, X=XbaI, S= SpeI and P= PstI. Please note: cutting with BsaI will result in an XbaI overhang.</figcaption></figure>At first, we removed a BsaI restriction enzyme site on the backbone of the vector by PCR based mutagenesis using primers TM3161 and TM3164 because it was interfering with our design. The confirmed BsaI free vector was then cut with EcoRI and XbaI to insert the Xylose inducible promoter P<sub>xylA</sub><sup>1</sup> wich was previously digested with EcoRI and BsaI (resulting in an XbaI overhang) to maintain the BioBrick prefix in front of the promoter. Next, we had to create an entirely new multiple cloning site (MCS): We synthesized a new RFP version using the original RFP derived from the pSB1C3 backbone. The expression of this RFPsyn2 still driven by the IPTG inducible P<sub>lacI</sub> promoter but it now lacks restriction enzyme sites which interfere with the RFC25 standard. Additionally, we added an AgeI restriction enzyme site in the BioBrick suffix which is necessary for translational fusions. Furthermore, we amplified a <i>lacZ</i> fragment with AgeI and NgoMIV restriction enzyme sites upstream of the coding sequence and the RFC10 BioBrick standard as suffix using the primers iG17P055 and iG17P056.
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<figcaption><b>Figure 1: New layout of the multiple cloning site in our Evaluation Vector.</b> The crosses indicate restriction enzyme sites: E= EcoRI, N= NotI, X=XbaI, S= SpeI and P= PstI. Please note: cutting with BsaI will result in an XbaI overhang.</figcaption></figure>At first, we removed a BsaI restriction enzyme site on the backbone of the vector by PCR based mutagenesis using primers TM3161 and TM3164 because it was interfering with our design. The confirmed BsaI free vector was then cut with EcoRI and XbaI to insert the Xylose inducible promoter P<sub>xylA</sub><sup>1</sup> wich was previously digested with EcoRI and BsaI (resulting in an XbaI overhang) to maintain the BioBrick prefix in front of the promoter. Next, we had to create an entirely new multiple cloning site (MCS): We synthesized a new RFP version using the original RFP derived from the pSB1C3 backbone. The expression of this RFPsyn2 still driven by the IPTG inducible P<sub>lacI</sub> promoter but it now lacks restriction enzyme sites which interfere with the RFC25 standard. Additionally, we added an AgeI restriction enzyme site in the BioBrick suffix which is necessary for translational fusions. Furthermore, we amplified a <i>lacZ&alpha;</i> fragment with AgeI and NgoMIV restriction enzyme sites upstream of the coding sequence and the RFC10 BioBrick standard as suffix using the primers iG17P055 and iG17P056.
 
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Revision as of 14:02, 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:

  1. Exchangeable promoter region
  2. Insertion of transcriptional and translational expression units
  3. Fulfilling the RFC10 and RFC25 BioBrick standard
  4. 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.

A scheme explaining the design of the Evaluation Vector.
Figure 1: New layout of the multiple cloning site in our Evaluation Vector. The crosses indicate restriction enzyme sites: E= EcoRI, N= NotI, X=XbaI, S= SpeI and P= PstI. Please note: cutting with BsaI will result in an XbaI overhang.
At first, we removed a BsaI restriction enzyme site on the backbone of the vector by PCR based mutagenesis using primers TM3161 and TM3164 because it was interfering with our design. The confirmed BsaI free vector was then cut with EcoRI and XbaI to insert the Xylose inducible promoter PxylA1 wich was previously digested with EcoRI and BsaI (resulting in an XbaI overhang) to maintain the BioBrick prefix in front of the promoter. Next, we had to create an entirely new multiple cloning site (MCS): We synthesized a new RFP version using the original RFP derived from the pSB1C3 backbone. The expression of this RFPsyn2 still driven by the IPTG inducible PlacI promoter but it now lacks restriction enzyme sites which interfere with the RFC25 standard. Additionally, we added an AgeI restriction enzyme site in the BioBrick suffix which is necessary for translational fusions. Furthermore, we amplified a lacZα fragment with AgeI and NgoMIV restriction enzyme sites upstream of the coding sequence and the RFC10 BioBrick standard as suffix using the primers iG17P055 and iG17P056.

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A scheme explaining the cloning of the Evaluation Vector.
Figure 2: Cloning scheme of the Evaluation Vector. The Evaluation Vector was created using a specific series of cloning experiments.
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Results

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