BBa_K2020042 - tRNA specific for tyrosine and UAG codon in E. coli

BBa_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 of the lacI regulated promoter BBa_R0010, this expression system can be induced by addition of IPTG while working with LB medium.

K2020043 – tRNA synthetase specific for the ncAA AzF and UAG codon in E. coli → AzF-RS

K2020044 – tRNA synthetase specific for the ncAA AminoY and UAG codon in E. coli → AminoY-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

K2020047 – tRNA synthetase specific for the ncAA 5HTP and UAG codon in E. coli → 5HTP-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 BBa_K792002)

K2020027 – leader sequence MFalpha (BBa_K2020026) plus fluorescence protein RFP (BBa_E2060)

K2020028 – leader sequence MFalpha (BBa_K2020026) plus fluorescence protein YFP (BBa_K165005)

K2020029 – leader sequence MFalpha (BBa_K2020026) plus fluorescence protein mOrange (BBa_E2050)

Screening System

K2020040 – screening plasmid for incorporation of non-canonical amino acids → pRXG (twin pFRY)

K2020041 – screening plasmid for incorporation of non-canonical amino acids → pRYG (twin pFRYC)

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 New 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”.

K2020044 – tRNA synthetase specific for the ncAA AminoY and UAG codon in E. coli → AminoY-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”.

K2020047 – tRNA synthetase specific for the ncAA 5HTP and UAG codon in E. coli → 5HTP-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”.

BBa_K2020052 to BBa_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”.