K2020042 - tRNA specific for tyrosine and UAG codon in E. coli

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 part BBa_K2020001 and the terminator BBa_B0010. BioBrick BBa_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 digest proteins, and simultaneously secret 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 induce 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 fulfilled a Polymerase Chain Reaction 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 in a colony with the correct sequence in opposition to our trial of gaining a positive clone while working with the first promoter BBa_R0011. For testing the activity of subtilisin E we let the cells grow on Skim Milk plates. These got clear when our sample was added, so the proteolytic activity of subtilisin E produced by our expression system could be proved.

K2020043 – tRNA synthetase specific for the ncAA AzF and UAG codon in E. coli → AzF-RS
K2020045 – tRNA synthetase specific for the ncAA NitroY and UAG codon in E. coli → NitroY-RS
K2020046 – tRNA synthetase specific for the ncAA CNF and UAG codon in E. coli → CNF-RS
K2020050 - tRNA synthetase specific for tyrosine and UAG codon in E. coli → Y-RS
K2020051 - mutated tRNA synthetase specific for tyrosine and UAG codon in E. coli → Y-RS with Y32G
K2020052 – tRNA synthetase specific for DMNB-serine and UAG codon in E. coli → version 1
K2020053 – tRNA synthetase specific for DMNB-serine and UAG codon in E. coli → version 2
K2020054 – tRNA synthetase specific for DMNB-serine and UAG codon in E. coli → version 3
K2020055 – tRNA synthetase specific for DMNB-serine and UAG codon in E. coli → version 4
K2020056 – tRNA synthetase specific for DMNB-serine and UAG codon in E. coli → version 5
K2020057 – tRNA synthetase specific for DMNB-serine and UAG codon in E. coli → version 6
K2020058 – tRNA synthetase specific for DMNB-serine and UAG codon in E. coli → version 7
K2020059 – tRNA synthetase specific for DMNB-serine and UAG codon in E. coli → version 8
K2020060 – tRNA synthetase specific for DMNB-serine and UAG codon in E. coli → version 9
K2020061 – tRNA synthetase specific for DMNB-serine and UAG codon in E. coli → version 10

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 digest 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.

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 has to hinder the organism in the expression of the protease while 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.

K2020002 – expression system for subtilisin E in E. coli
Details about this part can be seen above in the section “Best New Composite Part”.

Mutated Versions

K2020003 – mutated expression system for subtilisin E in E. coli (S221Y) The named BioBrick K2020002 above for the expression of subtilisin E in E. coli is the basic concept 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 part BBa_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 a site-directed mutagenesis. The codon AGC of serine²²¹ was substituted with TAC which codes for tyrosine.
By performing a site-directed mutagenesis, serine in the catalytic triade of the enzyme was exchanged against tyrosine. Therefore, the alkaline serine protease looses its proteolytic activity of non-specifically digesting proteins. Caused to its leader sequence pelB BBa_J32015, the BioBrick secrets an inactive version of subtilisin E into the periplasm of the cell as soon as it is introduced into E. coli.

K2020004 – mutated expression system for subtilisin E in E. coli (S221X)
K2020005 – mutated expression system for subtilisin E in E. coli (Y77W)
K2020006 – mutated expression system for subtilisin E in E. coli (Y77X)

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 one wants to be secreted. Therefore, 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.

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.

K2020041 – screening plasmid for incorporation of non-canonical amino acids → pRYG This part is an improvement of the existing BioBrick K1416003, called pFRYC by the iGEM Team Austin Texas 2014.

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”.

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”.

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”.

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”.

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”.

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”.

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”.