Difference between revisions of "Team:Bielefeld-CeBiTec/Results/translational system/library and selection"
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For the positive selection plasmids multiple approaches can be made. The classic approach is to us a resistence gene with amber codons. Therefore we provide the kanamycin resistance gene coding sequence (<a target="_blank" href=" http://parts.igem.org/Part:BBa_K2201409">BBa_K2201409</a>) 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 (<a target="_blank" href=" http://parts.igem.org/Part:BBa_K2201410">K2201410</a>) under the control of an araBAD promotor (<a target="_blank" href=" http://parts.igem.org/Part:BBa_K808000"> BBa_K808000</a>). <br><br> | For the positive selection plasmids multiple approaches can be made. The classic approach is to us a resistence gene with amber codons. Therefore we provide the kanamycin resistance gene coding sequence (<a target="_blank" href=" http://parts.igem.org/Part:BBa_K2201409">BBa_K2201409</a>) 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 (<a target="_blank" href=" http://parts.igem.org/Part:BBa_K2201410">K2201410</a>) under the control of an araBAD promotor (<a target="_blank" href=" http://parts.igem.org/Part:BBa_K808000"> BBa_K808000</a>). <br><br> | ||
− | <div class="figure | + | <div class="figure medium"> |
<img class="figure image" src="https://static.igem.org/mediawiki/2017/f/f0/T--Bielefeld-CeBiTec--pos-sel-res.png"> | <img class="figure image" src="https://static.igem.org/mediawiki/2017/f/f0/T--Bielefeld-CeBiTec--pos-sel-res.png"> | ||
<p class="figure subtitle"><b>Figure 11: Possible Positive Selection Plasmid based on a Resistance Gene.</b> The postive selection plasmid based on resistance containes the tRNA<sup>amber</sup> and the kanamycin resistance gene with two amber codons (S133Am and S154Am). The parts are shown here in pSB1C3.</p> | <p class="figure subtitle"><b>Figure 11: Possible Positive Selection Plasmid based on a Resistance Gene.</b> The postive selection plasmid based on resistance containes the tRNA<sup>amber</sup> and the kanamycin resistance gene with two amber codons (S133Am and S154Am). The parts are shown here in pSB1C3.</p> | ||
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Another approach for positive selection is a visual conformation. This can be achieved with a fluorescent protein. Based on Santoro <i>et al.</i> we provide a T7 RNA polymerase (T7 RNAP) and a GFP under control of the T7 promotor (Santoro et al., 2002). The T7 RNAP (<a target="_blank" href=" http://parts.igem.org/Part:BBa_K2201405">BBa_K2201405</a>) is under control of the inducible araBAD promotor (<a target="_blank" href=" http://parts.igem.org/Part:BBa_K808000"> BBa_K808000</a>). Also we provide the cosding sequence of T7 RNAP (<a target="_blank" href=" http://parts.igem.org/Part:BBa_K2201403">BBa_K2201403</a>) from the <i>E. coli </i> 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 (<a target="_blank" href=" http://parts.igem.org/Part:BBa_I746909">BBa_I746909</a>). We reversed (<a target="_blank" href=" http://parts.igem.org/Part:BBa_K2201404">BBa_K2201404</a>) this part so the probability of the GFP being expressed subliminally trough read through of the endogenous RNA polymerase sinks. <br><br> | Another approach for positive selection is a visual conformation. This can be achieved with a fluorescent protein. Based on Santoro <i>et al.</i> we provide a T7 RNA polymerase (T7 RNAP) and a GFP under control of the T7 promotor (Santoro et al., 2002). The T7 RNAP (<a target="_blank" href=" http://parts.igem.org/Part:BBa_K2201405">BBa_K2201405</a>) is under control of the inducible araBAD promotor (<a target="_blank" href=" http://parts.igem.org/Part:BBa_K808000"> BBa_K808000</a>). Also we provide the cosding sequence of T7 RNAP (<a target="_blank" href=" http://parts.igem.org/Part:BBa_K2201403">BBa_K2201403</a>) from the <i>E. coli </i> 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 (<a target="_blank" href=" http://parts.igem.org/Part:BBa_I746909">BBa_I746909</a>). We reversed (<a target="_blank" href=" http://parts.igem.org/Part:BBa_K2201404">BBa_K2201404</a>) this part so the probability of the GFP being expressed subliminally trough read through of the endogenous RNA polymerase sinks. <br><br> | ||
− | https://static.igem.org/mediawiki/2017/3/3a/T--Bielefeld-CeBiTec--pos-sel-flu.png | + | |
+ | <div class="figure medium"> | ||
+ | <img class="figure image" src="https://static.igem.org/mediawiki/2017/3/3a/T--Bielefeld-CeBiTec--pos-sel-flu.png"> | ||
+ | <p class="figure subtitle"><b>Figure 12: Possible Positive Selection Plasmid based on Fluorescence.</b> The postive selection plasmid based on fluorescence containes the tRNA<sup>amber</sup>, the T7 RNA polymerase with on amber codon and uvGFP under control of the T7 promotor. The parts are shown here in pSB1C3.</p> | ||
+ | </div> | ||
+ | |||
All parts for the positive selection could also be combined and used simultaneously (Liu and Schultz, 2010). <br><br> | All parts for the positive selection could also be combined and used simultaneously (Liu and Schultz, 2010). <br><br> | ||
− | https://static.igem.org/mediawiki/2017/f/fd/T--Bielefeld-CeBiTec--pos-sel-com.png | + | <div class="figure medium"> |
+ | <img class="figure image" src="https://static.igem.org/mediawiki/2017/f/fd/T--Bielefeld-CeBiTec--pos-sel-com.png"> | ||
+ | <p class="figure subtitle"><b>Figure 13: Possible Combined Positive Selection Plasmid based on a Resistance Gene and Fluorescence.</b> This postive selection plasmid containes the tRNA<sup>amber</sup>, the T7 RNA polymerase with on amber codon,uvGFP under control of the T7 promotor and the kanamycin resistance gene with two amber codons (S133Am and S154Am). The parts are shown here in pSB1C3.</p> | ||
+ | </div> | ||
+ | |||
+ | |||
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 (<a target="_blank" href=" http://parts.igem.org/Part:BBa_K2201406">BBa_K2201406</a>) (Liu and Schultz, 1999). We also combined the coding sequence with the inducible araBAD promotor (<a target="_blank" href=" http://parts.igem.org/Part:BBa_K808000"> BBa_K808000</a>) and reversed the whole sequence. <br><br> | 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 (<a target="_blank" href=" http://parts.igem.org/Part:BBa_K2201406">BBa_K2201406</a>) (Liu and Schultz, 1999). We also combined the coding sequence with the inducible araBAD promotor (<a target="_blank" href=" http://parts.igem.org/Part:BBa_K808000"> BBa_K808000</a>) and reversed the whole sequence. <br><br> | ||
− | https://static.igem.org/mediawiki/2017/2/26/T--Bielefeld-CeBiTec--neg-sel-olga.png | + | <div class="figure medium"> |
+ | <img class="figure image" src="https://static.igem.org/mediawiki/2017/2/26/T--Bielefeld-CeBiTec--neg-sel-olga.png"> | ||
+ | <p class="figure subtitle"><b>Figure 14: Possible Negative Selection Plasmid.</b> The negative selection plasmid containes the tRNA<sup>amber</sup> and barnase with two amber codons (Q4Am and D46Am). The barnase is under control of a araBAD promotor. The parts are shown here in pSB1C3.</p> | ||
+ | </div> | ||
+ | |||
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. <br><br> | 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. <br><br> | ||
Revision as of 17:51, 1 November 2017
Short Summary
Generating the Library
Figure 1: Library plasmid pSB1C3 containing the tyrosyl tRNA synthetase.
Library plasmid based on pSB1C3, containing the tyrosyl tRNA synthetase under controle of a glnS promotor, a pMB1 origin of replication and chloramphenicole resistance.
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.
Figure 3: Generating a synthetase library by using oligo dimers and mRFP as an optical controle.
Two primers, one with a randomized position, are designed to form a dimer (1), which is completed to dsDNA by the Klenow fragment. The region of the TyrRS meant to be modified by randomization is replaced by mRFP as an optical control (2). In the case oft he incorporation oft he randomized dsDNA, the mRFP is replaced and thus the incorporation is visible directly.
Figure 4: Tyrosyl-tRNA/synthetase library on LB-plate with chloramphenicol.
The library was generated by using two primers, one with a randomized position, which are designed to form a dimer. This dimer is completed to a dsDNA by the Klenow fragment. As optical control, a mRFP is incorporated in this certain position to be ranomized, which is then replaced by the dsDNA. On this basis, we could see that xxx% of the cells incorporated the template plasmid, but most cells contained the plasmid with the incorporated randomized dsDNA.
Anaylzing the Tyrosyl tRNA/Aminoacyl-Synthetase Library
Sequencing by Sanger
E(T)= n*Hn (1)
Figure 3: Chromatograms of the Tyr-RS Library by sanger sequencing.
Depicting the chromatograms of four TyrRS library replicates being sequenced forward and reverse by sanger sequencing. The positions 158, 159, 162 of the TyrRS are randomized by NNK scheme.
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.
Illumina Sequencing
Figure 4: Starting the Illumina MiSeq sequencing.
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.
The electropherogram of the Agilent BioAnalyzer High Sensitivity DNA Assay shows our amplified library fragment as the largest peak of 2,874.13 pg/µL and a molarity of 7,735.3 pmol/L with a length of 563 bp and flanked by the two markers (35 bp and 10,380 bp). The image of the gel, depicted in figure 5, shows a thick band at 550-600 bp, fading out up to the band of 700 bp, matching the slightly uneven peak of the electropherogram. It is important, that there are as less as possible larger fragments, forcing a possible overload and therefore the abruption of sequencing.
The MiSeq sequencing showed 1,650,024 reads with the NNK motif, consisting of 30,440 different variants. Determined by the rules of combinatorics, we calculated 32,768 possible sequence variants.
When analyzing the sequences for the NNN motif, we obtain 1,652,553 reads. The difference between the number of reads for the NNK motif to the NNN motif gives us 2,529 variants with a coverage of 1 and implies, that reads with a covering of 1 are misinterpreted. For this reason, we consider all 2,768 variants, showing coverage of 1, as possible false instances. When combining the possible false instances with the 30,400 different variants results in a total library size of 27,672 different sequence variants.
We identified, that reads with a coverage of minimal two result in 8,787 different peptides. 8,464 different peptides can be translated of the sequence variants with a coverage higher than two, and 8,135 with a coverage higher than three. Based on this data, our 27,672 variants composed tyrosyl tRNA/synthetase library codes for more than 8,000 different peptides.
Considering that we continued the generation of the library after sequencing, nearly doubling the number of clones, we assume the tyrosyl-tRNA/synthetase library to be larger than the analyzed 27,672 different sequences and 8,000 peptides. We were not allowed to submit the complete library.
Figure 5: Electropherogram of the tyrosyl- synthetase library.
The Agilent BioAnalyzer High Sensitivity DNA Assay is used for the measurement. The library fragments are depicted as the peak in the center (563 bp), flanked by markers.
Figure 6: Gel image of the Agilent BioAnalyzer High Sensitivity DNA Assay of the tyrosyl- synthetase library.
Therefore, we submitted two versions of the basis library Plasmid ( BBa_K2201400 , BBa_K2201411 ) for the generation of a own library. In addition to that, the complete library is available to all future iGEM teams, after request.
Selection
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.
Figure 7: Positive selection plasmid BBa_K2201900 .
Positive selection plasmid for the incorporation of ncAAs
The positive selection plasmid contains a tRNA and a kanamycin resistance with two amber codons. Cotransformed with the library of tRNA/aminoacyl-synthetase with random mutated binding sites, on kanamycin only the clones survive that could charge any amino acid to the tRNA in response to the UAG codon.
Figure 8: Negative selection plasmid Ba_K2201901
Negative selection plasmid against the incorporation of endogenous amino acids.
The negative selection plasmid contains an tRNA with the anticodon for the amber codon and a barnase containing amber codons at permissive sites. In the negative selection the target amino acid is not supplemented to the media. If the cotransformend clones from the positive selection charge endogenous amino acids to the tRNA, the cells die. This provides a selection method for high specific aaRS.
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.
Figure 9: Remaining colonies while the positive selection for 2-NPA, containing the positive selection plasmid and the library plasmid.
The remaining cells own an aaRS able to bind a non canonical or endogenous amino acid.
Figure 10: Remaining colonies while the negative selection and after the positive selection for 2-NPA, containing the neg selection plasmid and the library plasmid.
The remaining cells own an aaRS, specific to not bind an endogenous amino acid. Red colonies own a positive selection plasmid as result of the plasmid isolation and can be separated for further selection rounds easily.
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.
Outlook:Selection Process
But we also provided multiple basic parts to build selection plasmids due to individual needs. One part needed for both selection plasmids is the tRNAtyr (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).
Figure 11: Possible Positive Selection Plasmid based on a Resistance Gene. The postive selection plasmid based on resistance containes the tRNAamber and the kanamycin resistance gene with two amber codons (S133Am and S154Am). The parts are shown here in pSB1C3.
Figure 12: Possible Positive Selection Plasmid based on Fluorescence. The postive selection plasmid based on fluorescence containes the tRNAamber, the T7 RNA polymerase with on amber codon and uvGFP under control of the T7 promotor. The parts are shown here in pSB1C3.
Figure 13: Possible Combined Positive Selection Plasmid based on a Resistance Gene and Fluorescence. This postive selection plasmid containes the tRNAamber, the T7 RNA polymerase with on amber codon,uvGFP under control of the T7 promotor and the kanamycin resistance gene with two amber codons (S133Am and S154Am). The parts are shown here in pSB1C3.
Figure 14: Possible Negative Selection Plasmid. The negative selection plasmid containes the tRNAamber and barnase with two amber codons (Q4Am and D46Am). The barnase is under control of a araBAD promotor. The parts are shown here in pSB1C3.
References
Pfeufer V., Schulze M. , (2015). Laser fluorescence powers sequencing advances. BioOptica World Beese,L.S, Derbyshire V, Steitz T.A. (1993). Structure of DNA Polymerase I Kienow Fragment Bound to Duplex DNAMiddendorf L.R., Humpfrey P.G., Narayanan N., Roemer S.C.. (2008) Chapter8 Sequencing Technology. WILEY Guo, L.-T., Wang, Y.-S., Nakamura, A., Eiler, D., Kavran, J.M., Wong, M., Kiessling, L.L., Steitz, T.A., O’Donoghue, P., and Söll, D. (2014). Polyspecific pyrrolysyl-tRNA synthetases from directed evolution. Proc. Natl. Acad. Sci. U. S. A. 111: 16724–16729.
Liu, C.C. and Schultz, P.G. (2010). Adding New Chemistries to the Genetic Code. Annu. Rev. Biochem. 79: 413–444.
Liu, D.R. and Schultz, P.G. (1999). Progress toward the evolution of an organism with an expanded genetic code. Proc. Natl. Acad. Sci. 96: 4780–4785.
Santoro, S.W., Wang, L., Herberich, B., King, D.S., and Schultz, P.G. (2002). An efficient system for the evolution of aminoacyl-tRNA synthetase specificity. Nat. Biotechnol. 20: 1044–1048.
Umehara, T., Kim, J., Lee, S., Guo, L.-T., Söll, D., and Park, H.-S. (2012). N-Acetyl lysyl-tRNA synthetases evolved by a CcdB-based selection possess N-acetyl lysine specificity in vitro and in vivo. FEBS Lett. 586: 729–733.
Zhang, Y., Lamb, B.M., Feldman, A.W., Zhou, A.X., Lavergne, T., Li, L., and Romesberg, F.E. (2017). A semisynthetic organism engineered for the stable expansion of the genetic alphabet. Proc. Natl. Acad. Sci. 114: 1317–1322.