Revision as of 07:33, 11 September 2015

Expression System

The system we have designed relies on the presence of two Clickable Outer Membrane Proteins within the cells. To obtain cells which are functionalized with these proteins, we need an expression system capable of producing two proteins which carry non-natural amino acids. In addition to co-expression of two proteins, we thus need to gear up our bacteria with the tools to incorporate non-natural amino acids within their proteins. An overview of the expression system we used to obtain these bacteria is presented below.

Figure 1: As a proof of concept, we will construct and express our device within E.coli BL21DE3. The device we test features the fast cellular response signaling components. We will verify the construction of our device by conducting numerous experiments. These experiments include verification of the click reaction, of the individual signaling components, whether proximity invokes a response and if the aptamers work.
The BL21DE3 cells will be cotransformed with two plasmids. The first plasmid, pET-Duet1 (blue), carries the genes for the outer membrane proteins and expression is triggered by the addition of IPTG. The second plasmid, pEVOL pAzF (red), is necessary for the incorporation of the non-natural amino acid within the outer membrane proteins.

The Vectors

As shown in Figure 1, our system relies on the presence of two vectors within the host cell. The first of these vectors is pEVOL-pAzF. The second one is the pETDuet-1 expression vector.

pEVOL-pAzF

pEVOL is a small vector which has been designed and optimized for the incorporation of unnatural amino acids into proteins in E.coli. The coding sequence of pEVOL encodes tRNA synthetases, which can translate the amber stop codon sequence into the incorporation of the non-natural amino acid. Optimization of the vector has enabled higher yields of mutant proteins in comparison to previous vectors: pEVOL showed roughly 250% greater yields in comparison with vectors previously used for the incorporation of non-natural amino acids [1]. One of the first amino acids which has been incorporated into proteins using the relatively novel pEVOL vector was pAzF and we will use this exact vector to construct our mutant protein in vivo.

Features of the pEVOL vector:

The pEVOL expression vector features the p15A origin of replication which makes the pEVOL plasmid compatible
Plasmid compatibility is generally defined as the failure of two coresident plasmids to be stably inherited in the absence of external selection [2]. The cause of the failure to be stably co-inherited lies in the fact that the origins of replication are too analogous. In that case, the bacteria cannot distinguish between the plasmids and can eventually lose either one of the plasmids as the amount of both plasmids is limited by a single copy number.
with many other frequently used plasmids.
The chloramphenicol resistance gene
The tRNA synthetase is under the control of the arabinose-inducible AraBAD promotor

Figure 2: Simplified vectormap of the pAzF

pETDuet-1

The designed system relies on the two membrane proteins which come into close proximity as a result of ligand binding. Often, when such protein assemblies are to be obtained, one can isolate endogenous complexes and reconstitute those components in vitro to analyze whether the assembly takes place [3]. As we have designed the system to be used in vivo, however, we rely on the co-expression of all components within the same host cell.
Generally, this heterologous expression can be reached into two different ways, firstly by transforming multiple constructs and secondly by introducing a single plasmid carrying multiple genes in E.coli. As our device already featured two plasmids, we devised to use a plasmid which could co-express multiple genes, preferably in an equimolar ratio. Since the pETDuet-1^TM Expression System from Novagen has been developed for this particular purpose, we have chosen to use this system as our vector of choice.

Features of the pETDuet-1 expression vector:

The pET-Duet1 vector features two multiple cloning sites, each carrying a dozen cloning sites. This enables insertion of multiple fragments.
Each of the multiple cloning sites contains its own T7 lac promotor and a ribosome binding site. A single terminator is located after MCS2, such that transcription yields two different mRNAs.
The pET-Duet1 vector not only carries multiple genes but is bicistronic. Therefore, it allows simultaneous expression of two proteins separately from the same RNA transcript. Hence, both the MCS1 insert and MCS2 insert are expressed from the longer mRNA strand. Only the MCS2 insert is expressed from the shorter mRNA strand.
Figure A: Transcription of pETDuet-1 yields two different messenger RNA strands. The longer strain is bicistronic and encodes both the MCS1 as well as the MCS2 insert. The smaller strain only encodes MCS2. The smaller strain compensates for the reduced expression of MCS2 on the longer strand.

Figure 3: General overview of the pETDuet-1 expression vector. The vector carries the Ampicillin resistance gene and carries the pBR332 origin of replication

Usually, bicistronic vectors containing two target genes under the control of a single promotor preceding the two genes show strongly reduced expression of the gene located more distant from the promotor site [4]. The second promotor which initiates the translation of the second mRNA aims to correct for this reduced expression.

Non-Natural Amino Acid Expression

DNA is best known for its function as carrier of hereditary information: DNA carries the biological information that is carried over to the next generation each time cells divide into daughter cells. This biological information is merely composed of four base pairs, begin Guanine, Cytosine, Adenine and Tyrosine. The sequence of the basepairs is translated into a amino acid code in the form of triplets. These triplets are recognized by tRNA's which carry the correct to be incorporated amino acid which is added onto the growing chain (see Figure 4).

Figure 4: tRNAs play a key role in translation of the genetic instructions to amino acids. These tRNAs present the ribosomes with the correct amino acid for the correct codon. After the tRNAs recognize their corresponding codon, they enter the ribosomes where their amino acid will be added onto the growing peptide chain.

In addition to encoding a amino acid sequence, some of the triplets encode stop codons. In total, three of the 64 possible triplets encode these stop codons. Instead of a tRNA, the triplet is recognized by release factors which bind to the triplet and enter the ribosome. Since these release factors do not carry any amino acid, the amino acid chain will be released from the ribosome into the cytosol, allowing it to fold into intricate structures (see Figure 5).

All of the naturally occuring proteins are encoded by the 20 primary amino acids normally present in proteins. Since this severely limited protein research, researchers have explored the possibility of incorporating non-natural amino acids into proteins. A way to do this is to hijack the cells stop codon by introducing a non-natural tRNA to the cells. This tRNA will compete with the release factor for the stop codon and will incorporate the non-natural amino acid from time to time.