Difference between revisions of "Team:Bielefeld-CeBiTec/Results/translational system/library and selection"

The incorporation of a non‑canonical amino acid (ncAA) requires a tRNA/aminoacyl‑tRNA synthetase (tRNA/aaRS) pair which is able to accept and bind the ncAA (to charge the tRNA with the ncAA). Therefor aour aim is to generate a library of ncAAs with different binding sites. The aaRS, based on the wild type Methanococcus jannashii tyrosyl‑tRNA synthetase , is orthogonal to tRNA/aaRS from E.coli and suitable to incorporate novel ncAAs . To generate a library with random mutated amino acid binding sites we generated a template plasmid with an optical control and cloned the tyrosyl‑tRNA synthetase library, by using single stranded DNA annealing with randomized oligonucleotides. The library consist of approximately 150,000 library plasmids, including more than 27,672 variants of the tyrosyl‑tRNA synthetase. The tyrosyl‑tRNA synthetase library is the basis for positive and negative selection cycles , where an optimal adapted tyrosyl‑tRNA synthetase variant can be obtained.

For the generation of the tyrosyl-synthetase library, we used a method based on the dimerisation through the overlap of two randomized primers, which can be integrated on a certain position. The 3’ end of the two randomized primers face each other and overlap at the 3’ end. This homology, combined with the use of a Klenow Fragment is the key for this attempt. The Klenow Fragment is a purchasable Polymerase I fragment from Escherichia coli. It features a 5’-3’ Polymerase activity and a 3’-5’ Endonuklease activity (Beese et. al., 1993) and thereby can function as a polymerase to fill up smaller sequences based on a single stranded DNA. We use the Klenow Fragment to complete the dimer, generated from the primers, to a full double stranded sequence. This results in a small sequence which contains the randomized position and therefore filling the gap in the formed dimer by the use of the Klenow fragment. At this time, this formed small double stranded DNA fragment, containing the randomized position, can be integrated in the desired sequence by overlaps.
If a marker sequence is inserted in the position which should be randomized, the insertion of this certain sequence can be easily screened. We used a mRFP (BBa_J04450), under control of a lac promoter, lac operator and rrnB T1 terminator as an optical control. The primers 17vi and 17vj were designed with overlaps, homologous to the sequence around the binding pocket region synthetase sequence, allowing an optimal binding into the TyrRS. The position of the TyrRS chosen to be randomized, are Asp158, Ile159 and Leu162, the positions of the center of the binding pocket. The mRFP is inserted into the TyrRS in place of this binding side to function as an optical control. If the randomized DNA double strand is incorporated into the synthetase, the colonies color changes due to the absence of the mRFP, so the E. coli containing the randomized library plasmids can be easily picked for further positive and negative selection.

Generating a tyrosyl‑tRNA/synthetase (Tyr‑RS) library using the NNK scheme for the randomization of three positions of the binding pocket leads to a large variety of different sequences. When randomizing three codons by using the NNK scheme, there is a possibility of 32,768 different sequence variants. Considering the different apportionments of the codons coding for the same amino acid lead to 8,000 different possibilities of amino acid sequences, having influence on the structure of the binding pocket of the TyrRS. Determined by the rules of combinatorics, we calculated 32,768 possible sequence variants. Following the equation 1, using the free statistical software R (R coreteam 2015) we obtained a statistically needed library size of 393,447 randomized plasmids, so that every possible sequence variant occurs at least once.

E(T)= n*Hn (1)

We achieved a library size of more than 27,672 TyrRS plasmids. When analyzing our Tyr‑RS library with the sanger sequencing, an unexpected signal distribution of the nucleotides occurred: Using certain fluorophores for the labeling in sanger sequencing, the fluorescence signal of the different labeled nucleotides is not identical.

First, the different signal intensities seemed to be caused by the use of the labelling fluorophores in sanger sequencing. In general, when using four-colour labelling on the dNTPs, the signal-to-noice ratio is reduced because of the spectral overlap of the fluorescence emission. This results in shorter and less accurate reads (Middendorf et.al.,2008). In addition to that, the excitation with one single wavelength compromises the sequencing results. That is due to the wide range of the varying absorption and fluorescence emission spectra (500‑800 nm) of the used fluorophores by a laser excited fluorescence of approximately 488 nm (Pfeufer et.al.,2015).

Regarding the chromatograms, depicted in Figure 4, the maximal fluorescence intensity of the thymidine is approximately 75 % up to 90 % lower than the maximal fluorescence intensity of the guanine. In comparison, the maximal fluorescence signal of the guanosine shows up to 97 % of the approximate maximal fluorescence signal of the cytosine.

Comparing these tendencies with the sequence results of the modified positions (NNK), lead to the following assumption: when generating a library using the NNK scheme, the rates for the incorporation of the different nucleotides is not evenly distributed. Originally, we expected an equal distribution of guanosine and thymidine on the K position, but the fluorescence signal of the thymidine is approximately higher than the fluorescence signal of the guanosine. Despite the given data of the maximal fluorescence intensity of the four nucleotides, at this position, the fluorescence signal of the thymidine is higher than the guanosines signal. This implies a higher incorporation rate of the thymidine if the sequence is randomized with a K in this position. Analogue to this experience, the distribution of the incorporation of the four nucleotides, resulting from an N randomization on this certain position, is not equal either. In relation to the other three incorporated nucleotides, there is an approximately higher cytosine signal on this position, also implying a higher incorporation rate of cytosine when using the N randomization.

We generated oligonucleotides containing a certain adapter sequence and a unique indice to separate our sequences from other libraries. The amplified region of our library had a maximal length of 500 bp, so the DNA fragments do not get entangled while bridge amplification, which would lead to an overlap of the different clusters. After amplification with the certain oligonucleotides, the PCR product was purified from a 1 % agarose gel. The quality of the library amplificate was controlled for the NGS by using the Agilent BioAnalyzer with High-Sensitivity DNA chip. This technology uses capillary electrophoresis for a sensitive quantification and sizing of DNA fragments to test if our library preparation matches the specifications of the Illumina MiSeq technology.

Screening the whole library for its ability to incorporate the desired ncAAs specific would be too time-consuming. We decided to create a high throughput method for the selection of the clones that incorporate the target ncAA. The selection system is based on two selection steps that have to be repeated several times. In the first step, the positive selection, all clones that incorporate amino acids in response to the amber codon survive. The second step, the negative selection, is to select for specifity of the tyrosyl-tRNA synthetase. For both selection steps the library is cotransformed with the selection plasmid in pSB3T5 to prevent incompatibility to the library plasmid in pSB1C3. The plasmid charts of the selection plasmids are shown below as BioBricks.

The positive selection plasmid (BBa_K2201900) contains the Methanococcus jannaschii based tRNA (CUA) with an anticodon for the amber codon under the constitutive promoter proK. The essential part for the selection is the kanamycin resistance with two amber codons behind the translation start. If the tRNA/aminoacyl-synthetase mutant (encoded on a cotransformed library plasmid is able to charge the tRNA (CUA) with any amino acid) the cell could express the kanamycin resistance. Thus, these cells survive when plated out at LB agar plates with the ncAA and kanamycin.

In the negative selection only the cells that specific incorporate the ncAA, and not any endogenous amino acid, should survive. Therefore, the negative selection plasmid (BBa_K2201901) contains a toxin for E. coli, the barnase. Two amber codons are incorporated at permissive sites of the barnase and the plasmid contains the same tRNA (CUA) as the positive selection plasmid. In contrast to the positive selection, the cells are plated out on agar not containing the ncAA. Thus only synthetases charge the tRNA (CUA) which charge the tRNA with endogenous amino acids. These cells express the barnase and die.

Our goal is to generate a tRNA/synthetase which is able to incorporate 2-Nitro-L-phnylalanine, used for the photocleaving of the polypeptide backbone.

For the first round of selection, we cotransformed the library plasmid BBa_K2201400 with the (BBa_K2201900) and cultivated the cells on LB-plates with kanamycin and 2-nitrophenylalanine (2-NPA). Due to the amber stop codon, integrated in the kanamycine resistance on the positive selection plasmid, only the cells owning a functional aaRS survive. That is due to the amber stop codon on the kanamycin resistance. The resistance is only expressed, when a non canonical or endogenous amino acid is incorporated as response to the amber stop codon. Thus, the aaRS are selected for its function. To avoid an additional pressure on the cells, we did not used tetracycline or chloramphenicol for the cultivation, due to the dependency of the kanamycin expression on the library plasmid and the positive selection plasmid.

After the positive selection, we received approximately 800 colonies, showing that many of our generated TyrRS variants are able to bind a non canonical or endogenous amino acid despite the modifications. We washed these colonies off the plates, isolated the plasmids and cotransformed them with the negative selection plasmid (BBa_K2201901) and cultivated the cells on LB-plates with tetracycline and chloramphenicol to be certain to attain both plasmids.

Only cells, owning an aaRS which is as specifically that it does not bind an endogenous amino acid survived, due to the expression the barnase when responding to the amber stop codon and therefore binding of an endogenous amino acid. From the negative selection, we received < 100 colonies, showing a loss of more than 80 % of the aaRS candidates with which we first started the positive selection.
We combined the positive selection plasmid with a strengthening system (BBa_K2201373) , containing a T3 RNA-Polymerase with a reversed mRFP under T3 RNA-polymerase. With this system, the mRFP is expressed, resulting in a red colour of the colonies, still owning this positive selection plasmid. Thereby, it was possible to easily identify the clones owning the positive and not the negative selection plasmid while the negative selction. As it can be seen in Figure 10, the transformation efficiency of the positive selection plasmids, in contrast to the library plasmids, is low, resulting in one single false colony owning the positive selection plasmid.

After generating a synthetase library, selection process need to be performed to acquire library mutants with the highest specifity to a given non-canonical amino acid and still intact orthogonality to its corresponding tRNA. This selection is performed with positive and negative selection plasmids with an antibiotic resistence and a toxic gene with amber stop codons, respectively. We used a kanamycin resistence gene for the positive selection plasmid (BBa_K2201900) and a barnase gene for the negative selection plasmid (BBa_K2201901). Both plasmids had a low copy origin of replication (pSB3T5).

But we also provided multiple basic parts to build selection plasmids due to individual needs. One part needed for both selection plasmids is the tRNA^tyr (BBa_K2201408). It is the orthogonal tRNA pair to the synthetase library (BBa_K2201409) without any amber codons. This coding sequence can be adapted to be used with the promotor of choice and the amber codons at a desired location. Futhermore we provide the kanamycin resistance with two amber codons (K2201410) under the control of an araBAD promotor ( BBa_K808000).

Another approach for positive selection is a visual conformation. This can be achieved with a fluorescent protein. Based on Santoro et al. we provide a T7 RNA polymerase (T7 RNAP) and a GFP under control of the T7 promotor (Santoro et al., 2002). The T7 RNAP (BBa_K2201405) is under control of the inducible araBAD promotor ( BBa_K808000). Also we provide the cosding sequence of T7 RNAP (BBa_K2201403) from the E. coli KRX strain without amber codons to be adapted as needed. The needed GFP under the T7 promotor can already be found in the parts reg (BBa_I746909). We reversed (BBa_K2201404) this part so the probability of the GFP being expressed subliminally trough read through of the endogenous RNA polymerase sinks.

All parts for the positive selection could also be combined and used simultaneously (Liu and Schultz, 2010).

For the negative selection plasmid we just provide barnase as a toxic gene due to ccdB not being allowed anymore in the iGEM competition (Umehara et al., 2012). The coding sequence of barnase contains two amber codons (BBa_K2201406) (Liu and Schultz, 1999). We also combined the coding sequence with the inducible araBAD promotor ( BBa_K808000) and reversed the whole sequence.

Interestingly the question was raised if the library selection is influenced by the used selection plasmids and selection protocols (Umehara et al., 2012; Guo et al., 2014). Recently the Romesberg lab could also show, that a combination between positive selection and deep sequencing was enough to yield functional and specific synthetases (Zhang et al., 2017). Further research in this area might provide compelling results.