K2020042 - tRNA specific for tyrosine and UAG codon in E. coli For incorporating non-canonical amino acids into a protein, an orthogonal tRNA/ synthetase pair is needed, that does not crossreact with the cognate tRNA/ synthetase pairs. This tRNA can be assembled with a variety of synthetases into a plasmid to incorporate ncAA in E. coli in response to an amber stop codon.
This tRNA derives from the wild type tyrosyl Methanococcus janaschii tRNA/ synthetase pair. It was proven to not crossreact with the cognate E. coli tRNA/ synthetase pairs. The tRNA is used together with a tRNA-Synthetase. It has been proven to work with various synthetases for incorporation of ncAA by iGEM Team Austin Texas 2014

1. Y-RS, canonical amino acid
2. ONBY-RS
3. AzF-RS
4. CNF-RS
5. IodoY-RS
6. 5HTP-RS
7. NitroY-RS
8. AminoY-RS

Furthermore, iGEM Team TU Darmstadt was working with this tRNA and OMeY-synthetase.
Our Team is using the tRNA in this project to incorporate the canonical amino acid tyrosine with Y-RS in response to an amber stop codon as well as ONBY with ONBY-RS and in E. coli. Furthermore, DMNBS synthetase clones undergo a screening for incorporation efficiency and fidelity, which are done by using this BioBrick. These clones are proven to work with the tRNA and are all listed in the section Parts Collection.

Incorporation of ncAA:
The tRNA contains an amber anticodon for incorporating the ncAA in response to a recoded amber termination codon. It has been used previously in amberless E.coli strain C321.∆A.expb as well as BL21 DE3 gold. When working with a recoded amber codon in BL21 DE3, the tRNA is competing with release factor1 at the amber stop codon. As amber stop codons add up to only about 7% of total stop codos, amber suppression still works appropriately. Application of the tRNA is either the incorporation of the ncAA into a protein or usage with a reporter plasmid for example pFRY for probing ncAA tRNA/ synthetase pair clones regarding efficiency and fidelity.

Assembly in a synthetase plasmid for incorporation of ncAA:

Most synthetases are used with low copy plasmids (e.g. pACYC). Assemble the tRNA and the synthetase into a low copy plasmid, each one with an own promoter and one terminator for both (fig. 1). If your application is not for incorporation into a protein but the use with a second plasmid, make shure to use replicons from different incompatibility groups, eg. ColE1 and p15A and different selection markers.
All details about this part can be seen on its registry page.

Elements of orthogonality:

1. C1-G72; most important element for orthogonality. Recognised by Arg174, Arg132, Met178, Lys175 within the synthetase [2]
2. A73; recognised by Val195 [2]
3. G71; recognised by Arg132 [2]

Recognition between tRNA and ncAA-synthetases:
Methanococcus janaschii wild type tyrosyl tRNA consists of two arms: Firstly the acceptor-minihelix, where the ncAA will be attached to the 3' end. Secondly the anticodon containg arm. Synthetases interact mainly with the acceptor minihelix of the tRNA. Due to the lack of most of a recognizing element within the anticodon containg section, a mutation of a anticodon base has a relatively small effect on the aminoacylation efficiency [2] and may explain why a variety of ncAA can be incorporated with this tRNA.

Example measurement proving incorporation of amino acids:

Wild Type Methanococcus janaschii tRNA/synthetase pair for incorporation of tyrosine at an amber termination codon - the pair containing this tRNA - is cotransformed with pFRY - Flourescent reporter plasmid for measurement of incorporation of ncAA into BL21 DE3 gold. Reporter plasmid is induced by 100 µM ITPG.
pFRY is one part of a two plasmid reporter system for measurement of incorporation of ncAA via amber supression. It consists of two fluorescent domains connected through a linker sequence containing and amber stop. When IPTG induced and expressed, the fluorescence intensity can be measured. A red fluorescence is always visible upon induction, and if an amino acid is incorporated as response to the recoded amber stop codon, then a green fluorescence intensity is measurable. Fidelty and efficiancy of the incorporation can be determined with comparison of fluorescent level.
This experiment is performed in order to obtain fluorescence spectra of mRFP1 and sfGFP. As you can see in fig.3 GFP formation was measured as a result from successful amino acid incorporation via amber supression.
Excitation and emission spectra of mRFP1 (fig 2), and sfGFP (fig 3).were obtained from measurement with a modified Biolector set up.

K2020002 – expression system for subtilisin E in E. coli This expression system consists of the promoter BBa_R0011, the ribosome binding site BBa_B0034, the newly created BioBrick K2020001 and the terminator BBa_B0010. BioBrick K2020001 is a composite part itself and includes the secretion tag pelB (BBa_J32015) and a subtilisin E gene optimized for Escherichia coli codon usage (BBa_K2020000). Once introduced into E. coli, this BioBrick is able to produce subtilisin E, an alkaline serine protease, which non-specifically digests proteins, and simultaneously secrets the enzyme into the periplasm of the cell. Caused by the lacI regulated promoter BBa_R0010, the expression system can be induced by addition of IPTG.
With the iGEM promoter BBa_R0011, which was integrated in our sequence at first, it was not possible to successfully express subtilisin E due to fatal mutations inside the expression system in all analyzed colonies. Either there have been single base deletions or insertions in the pro-peptide, which led to a frameshift of the whole protein, or a 23 base pair deletion in the promoter. Both types of mutations result in an incorrect expression system, so that an expression of the protease is impossible. Since the promoter BBa_R0011 is leaky and induces the expression even without addition of IPTG, it can be assumed that subtilisin E is toxic for E. coli.
Hence, we exchanged the promoter against BBa_R0010. For achieving this, we carried out a polymerase chain reaction (PCR) to extract everything but the promoter and the RBS and simultaneously extend the remaining DNA sequence with the pre-fix of iGEM. Afterwards, we assembled it with the BioBrick J04500 and in parallel cloned it into the vector pSB1C3 - by cutting RFP out of the BioBrick J04450. The implemented BioBrick J04500 itself contains another IPTG inducible promoter (BBa_R0010) and the same RBS (BBa_B0034). An expression with the newly integrated promoter BBa_R0010 led to a colony with the correct sequence in opposition to our trial of gaining a positive clone while working with the first promoter BBa_R0011.
We continued our experiments by performing a skim milk assay on agar plates. Therefore, we poured LB skim milk agar plates containing IPTG and the needed antibiotic and streaked out the E. coli BL21 cells containing the plasmid with the expression system.

Comparing the clearance of the skim milk plates, a proteolytic activity could be proven for the cells containing the expression system for native subtilisin E. As a result, we concluded that within three days these cells are able to produce the native protease, which will then digest the skim milk in the agar plates, resulting in a clearance.
In conclusion, we were able to express subtilisin E in E. coli and to prove its proteolytic activity via skim milk assay.
All details about this part can be seen on its registry page.

K2020043 – tRNA synthetase specific for the ncAA AzF and UAG codon in E. coli → AzF-RS All details about this part can be seen on its registry page.

K2020045 – tRNA synthetase specific for the ncAA NitroY and UAG codon in E. coli → NitroY-RS All details about this part can be seen on its registry page.

K2020046 – tRNA synthetase specific for the ncAA CNF and UAG codon in E. coli → CNF-RS All details about this part can be seen on its registry page.

K2020050 - tRNA synthetase specific for tyrosine and UAG codon in E. coli → Y-RS All details about this part can be seen on its registry page.

K2020051 - mutated tRNA synthetase specific for tyrosine and UAG codon in E. coli → Y-RS with Y32G All details about this part can be seen on its registry page.

K2020052 – tRNA synthetase specific for DMNB-serine and UAG codon in E. coli → version 1 All details about this part can be seen on its registry page.

K2020053 – tRNA synthetase specific for DMNB-serine and UAG codon in E. coli → version 2 All details about this part can be seen on its registry page.

K2020054 – tRNA synthetase specific for DMNB-serine and UAG codon in E. coli → version 3 All details about this part can be seen on its registry page.

K2020055 – tRNA synthetase specific for DMNB-serine and UAG codon in E. coli → version 4 All details about this part can be seen on its registry page.

K2020056 – tRNA synthetase specific for DMNB-serine and UAG codon in E. coli → version 5 All details about this part can be seen on its registry page.

K2020057 – tRNA synthetase specific for DMNB-serine and UAG codon in E. coli → version 6 All details about this part can be seen on its registry page.

K2020058 – tRNA synthetase specific for DMNB-serine and UAG codon in E. coli → version 7 All details about this part can be seen on its registry page.

K2020059 – tRNA synthetase specific for DMNB-serine and UAG codon in E. coli → version 8 All details about this part can be seen on its registry page.

K2020060 – tRNA synthetase specific for DMNB-serine and UAG codon in E. coli → version 9 All details about this part can be seen on its registry page.

K2020061 – tRNA synthetase specific for DMNB-serine and UAG codon in E. coli → version 10 All details about this part can be seen on its registry page.

Basic Building Blocks

K2020000 – subtilisin E gene, optimized for E. coli codon usage The gene of this BioBrick can be used to express subtilisin E, which is an alkaline serine protease, which non-specifically digests proteins, in Escherichia coli. To use this part a suitable leader sequence has to be placed in front as the sequence of this BioBrick does not contain a start codon. Its sequence was originally obtained from a wild type Bacillus subtilis but was codon optimized for E. coli.
All details about this part can be seen on its registry page and in the section of the related BioBrick K2020002.

K2020001 – subtilisin E gene, optimized for E. coli codon usage, with leader sequence pelB This composite part consists of the leader sequence pelB (BBa_J32015) and the subtilisin E gene (K2020000) from our first BioBrick. It can be used to express subtilisin E in Escherichia coli and simultaneously secret the enzyme into the periplasm of the cell. Subtilisin E is an alkaline serine protease, which non-specifically digests proteins.
To generate a functional coding sequence that can be expressed in E. coli the leader sequence pelB, which begins with a start codon, was placed in front of the subtilisin E gene.
Caused to subtilisin E’s partly toxicity for E. coli, this BioBrick should be cloned into an expression system with an inducible promoter. This should hinder the organism in the expression of the protease during its growing period. With the iGEM promoter BBa_R0011 it was not possible to successfully express subtilisin E. Hence, we exchange the promoter against BBa_R0010.
All details about this part can be seen on its registry page and in the section of the related BioBrick K2020002.

K2020002 – expression system for subtilisin E in E. coli
Details about this part can be seen above in the section Best New Composite Part and on its registry page.

Mutated Versions

K2020003 – mutated expression system for subtilisin E in E. coli (S221Y) The BioBrick named K2020002 for the expression of subtilisin E in E. coli is the basic component of this new part. Therefore, it consists of the promoter BBa_R0010, the ribosome binding site BBa_B0034, the leader sequence pelB BBa_J32015, the newly created BioBrick K2020000 and the terminator BBa_B0010 like the expression system itself. The whole expression is based on the usage in E. coli, so the sequence of the subtilisin E gene from a wild type Bacillus subtilis was optimized for E. coli codon usage.
The sequence was partly ordered from IDT (BBa_K2020001 + BBa_B0010) and then cloned into BBa_J04500, a protein expression backbone, which already carries the LacI promoter BBa_R0010 and the ribosome binding site BBa_B0034. Afterwards, a mutation in the active site of the enzyme was introduced by performing site-directed mutagenesis (SDM). The codon AGC of serine²²¹ was substituted with TAC, which codes for tyrosine, so serine was exchanged against tyrosine in the catalytic triade of the enzyme.
We were not able to exactly detect the expression of the modified proteases via SDS gel, so we proceeded by executing a skim milk assay on agar plates containing IPTG and the needed antibiotics. Therefore, we streaked out the cells containing the modified expression systems on these plates and incubated at 30°C for three days.

Neither the empty backbone nor the SDM 1 modified expression system did seem to cause a proteolytic activity. A clearance and therefore a proteolytic activity could only be observed for the native protease (as demonstrated in the section of BioBrick K2020002). By demonstrating that this modification doesn’t result in a clearance of the skim milk plates, we were now able to prove that serine is essential for the proteolytic activity of the protease and that exchanging it would inactivate the enzyme. Hence, we demonstrated that exchanging serine against a photo-labile, non-canonical amino acid will inactivate subtilisin E and therefore proved the principle of our project.
All details about this part can be seen on its registry page.

K2020004 – mutated expression system for subtilisin E in E. coli (S221X) This part is a variation of the BioBrick K2020003. In opposition to the exchange of serine²²¹ against tyrosine, the named serine was exchanged against an amber codon, the least used stop codon in Escherichia coli. The codon AGC of serine²²¹ was substituted with TAG, which codes for DMNB-serine, if the corresponding aminoacyl tRNA/ synthetase pair is added. So serine will be exchanged against DMNBS in the catalytic triade of the enzyme in the presence of the needed DMNBS tRNA/ synthetase pair. By irradiation with light, this non-canonical amino acid can be converted to natural serine in the active site of the enzyme and the cleaved off photo-labile group. Thereby, the protease will reach its activity just by shining light on it and will be reversibly inactivated successfully.
Unfortunately, we were not able to execute all of the experiments that we planned due to a lack of time. But as our proof of principle of exchanging serine against a bigger amino acid namely tyrosine worked, it can be assumed, that this part will also operate correctly.
All details about this part can be seen on its registry page.

K2020005 – mutated expression system for subtilisin E in E. coli (Y77W) The BioBrick named K2020002 for the expression of subtilisin E in E. coli is the basic component of this new part. Therefore, it consists of the promoter BBa_R0010, the ribosome binding site BBa_B0034, the leader sequence pelB BBa_J32015, the newly created BioBrick K2020000 and the terminator BBa_B0010 like the expression system itself. The whole expression is based on the usage in E. coli, so the sequence of the subtilisin E gene from a wild type Bacillus subtilis was optimized for E. coli codon usage.
The sequence was partly ordered from IDT (BBa_K2020001 + BBa_B0010) and then cloned into BBa_J04500, a protein expression backbone which already carries the LacI promoter BBa_R0010 and the ribosome binding site BBa_B0034. Afterwards, a mutation between the pro-peptide and the subtilisin E gene was introduced by performing site-directed mutagenesis. The codon TAT of tyrosine⁷⁷ was substituted with TGG, which codes for tryptophan, so tyrosine was exchanged against tryptophan at the connection site of pro-peptide and subtilisin E gene. Caused by the mutation in the pro-peptide and not in the direct subtilisin E gene, the actual position of the modified tyrosine was counted from the beginning of the pro-peptide compared to common annotations counted from the N-terminus of the actual enzyme.
We were not able to exactly detect the expression of the modified proteases via SDS gel, so we proceeded by executing a skim milk assay on agar plates containing IPTG and the needed antibiotics. Therefore, we streaked out the cells containing the modified expression systems on these plates and incubated at 30°C for three days.

The empty backbone didn't cause a proteolytic activity. A clearance and therefore a proteolytic activity could be observed for the native protease (as demonstrated in the section of BioBrick K2020002) but also for the SDM 3 modified expression system. As SDM 3 had been executed to exchange tyrosine in the pro-peptide cleavage site against tryptophan, a proteolytic activity could be assumed caused by the clearance observed. Contrary to our former beliefs, it could now be deduced that exchanging tyrosine doesn’t result in a change of activity. Consequently, tyrosine in the pro-peptide cleavage site is not essential for the activity of subtilisin E.
Unfortunately, we were not able to prove the principle of our project regarding tyrosine in the pro-peptide cleavage site, as it seems to be not essential for the proteolytic activity. Thus, exchanging tyrosine against a photo-labile, non-canonical amino acid more precisely ONB-tyrosine will not influence the activity of the enzyme.
All details about this part can be seen on its registry page.

K2020006 – mutated expression system for subtilisin E in E. coli (Y77X) This part is a variation of the BioBrick K2020005. In opposition to the exchange of tyrosine⁷⁷ against tryptophan, the named tyrosine was exchanged against an amber codon, the least used stop codon in Escherichia coli. The codon TAT of tyrosine⁷⁷ was substituted with TAG, which codes for ONB-tyrosine, if the corresponding tRNA/ synthetase pair is added. So tyrosine will be exchanged against ONBY in the pro-peptide cleavage site of the enzyme in the presence of the needed ONBY tRNA/ synthetase pair. By irradiation with light, this non-canonical amino acid can be converted to natural tyrosine in the pro-peptide cleavage site of the enzyme and the cleaved off photo-labile group. Thereby, the protease will reach its activity just by shining light on it. Caused by the mutation in the pro-peptide and not in the direct subtilisin E gene, the actual position of the modified tyrosine was counted from the beginning of the pro-peptide compared to common annotations counted from the N-terminus of the actual enzyme.
Unfortunately, we were not able to execute all of the experiments that we planned due to a lack of time. But as our proof of principle of exchanging tyrosine against a bigger amino acid namely tryptophan showed no difference in the activity compared to our working subtilisin E expression system, tyrosine⁷⁷ in general not influences the proteolytic activity of the enzyme. And the replacement of tyrosine against ONBY will not cause an inactivation of the protease.
All details about this part can be seen on its registry page.

Improvement of an Existing Part

K2020026 – leader sequence MFalpha (different version of the biobrick K792002) This part is a different version of the leader sequence MFalpha (BBa_K792002) for Saccharomyces cerevisiae. It can be cloned directly in front of the protein, which will be secreted. It will be relocated into the medium and the tag will be cleaved off during this process. This alpha mating factors secretion tag is naturally occurring in the genome of S. cerevisiae. Futhermore, the sequence contained an illegal restriction site, which was corrected via PCR by us.
All details about this part can be seen on its registry page.

Screening System

K2020040 – screening plasmid for incorporation of non-canonical amino acids → pRXG This part is an improvement of the existing BioBrick K1416004, called pFRY by the iGEM Team Austin Texas 2014.
All details about this part can be seen on its registry page.

tRNA and Synthetases

K2020042 – tRNA specific for tyrosine and UAG codon in E. coli
Details about this part can be seen above in the section Best Basic Part and on its registry page.

K2020043 – tRNA synthetase specific for the ncAA AzF and UAG codon in E. coli → AzF-RS
Details about this part can be seen above in the section Parts Collection and on its registry page.

K2020045 – tRNA synthetase specific for the ncAA NitroY and UAG codon in E. coli → NitroY-RS
Details about this part can be seen above in the section Parts Collection and on its registry page.

K2020046 – tRNA synthetase specific for the ncAA CNF and UAG codon in E. coli → CNF-RS
Details about this part can be seen above in the section Parts Collection and on its registry page.

K2020050 – tRNA synthetase specific for tyrosine and UAG codon in E. coli → Y-RS
Details about this part can be seen above in the section Parts Collection and on its registry page.

K2020051 – mutated tRNA synthetase specific for tyrosine and UAG codon in E. coli → Y-RS with Y32G
Details about this part can be seen above in the section Parts Collection and on its registry page.

K2020052 to K2020061 - tRNA synthetase specific for DMNB-serine and UAG codon in E. coli → version 1 to 10
Details about these parts can be seen above in the section Parts Collection and on their registry pages.