RBS_library¶

RBS_library: Create a library of different RBS expression levels.

Options and default values:

• target=5000000 Target expression level to reach for in AU. If target is reached, computation is stopped, and library will be created. if target is not reached within the specified number of mutations, a library of expression levels closest to target as possible will be created.
• n=10 Number of library sequences to create.
• max_mutations=10 Maximum number of mutations to attempt.
• method=exp How to create the library. Two methods are available: exp and fuzzy. exp creates a library where each new sequence is an m-fold improvement over the last. m can either be supplied via the m-parameter, or calculated automatically to create an evenly spaced library from wt level to target. The exp method runs multiple Monte Carlo simulations to reach each target, however, it uses information from previous runs to more quickly reach subsequent targets. fuzzy tries to replicate the exp library, only the Monte Carlo simulation is only run once, and inbetween are collected along the way. This method yields a less precise library, but is quicker. Additionally, fuzzy enables picking out the best sequences below a certain mutation count by using the m parameter. Fx. using m=6, fuzzy will collect the best possible sequences with a maximum of 1, 2, .. 6 mutations. It will then try to fill out the rest of the library with evenly spaced sequences.
• m=0 see method for explanation on m.

This operation will run an Monte-Carlo simulation in an attempt to reach the specified target value within a number of mutations. Lower numbers of mutations are tried first and are always prioritised over similar expression levels with a higher mutation count.

translational_knockout¶

translational_knockout: Gene knock-out by premature stop-codon.

Tries to knock out a gene by introducing a number of early stop-codons in the CDS with the least amount of mutations possible.

Options and default values:

• ko_frame=0: Number of codons that are applicable to be mutated. E.g. a value of 10 means the operation will try to mutate stop codons into the CDS within 10 codons of the start codon. The default of 0 is within one half of the length of the CDS.
• ko_mutations=3: number of stop codons to introduce. Default (and minimum) is the number of different stop codons available in the genome configuration file (normally 3).

start_codon_optimal¶

start_codon_optimal: Mutates a start codon to the optimal start codon.

Tries to mutate the start codon to the optimal start codon defined in the strain config file (Usually ATG).

Options and default values:

• None

deletion¶

deletion: Delete nucleotides.

Options:

• delete: What to delete. An integer (number of nucleotides) or a string of nucleotides.
• position: Position of deletion.

Delete the nucleotides specified in delete from a DNA sequence.

Delete AAC from position 13 in thiD:

thiD deletion delete=AAC,position=13

Delete 3 nucleotides from position 13 in thiD:

thiD deletion delete=3,position=13

Specifying nucleotides to delete enables error-checking, but when deleting large stretches, an integer is more handy.

insertion¶

insertion: Insert nucleotides.

Options:

• insert: String of nucleotides to insert.
• position: Insertion position.

Examples:

thiD insertion insert=TTT,position=7
#Insert TTT as position 7 in thiD.

Sequence before is ATGAAACGA, and after insertion it is ATGAAAtttCGA.

find_mutation¶

find_mutation: Automatically find a mutation in a sequence.

Options:

• mutation=UPSTREAM[BEFORE->AFTER]DOWNSTREAM

Example:

rpoD find_mutation mutation=GAGCAA[AAC->TAG]CCG

Finds GAGCAAAACCCG in the sequence an changes it to GAGCAATAGCCG by creating the mutation AAC->TAG at position 10.

mutation¶

mutation: Any kind of nucleotide substitution.

Options:

• position: Position of first nucleotide.
• mutation=BEFORE->AFTER: mutate BEFORE to AFTER in sequence.

Substitute BEFORE nucleotides in sequence to AFTER at position.

Example sequence: ATGGCTGAA.

Mutate GCT to AAA at position 4:

araC mutation mutation=GCT->AAA,position=4
#Sequence after mutation: ATGaaaGAA.

Insert AAA as position 4:

araC mutation mutation=->AAA,position=4
#Sequence after mutation: ATGaaaGCTGAA.

Delete GCT at position 4:

araC mutation mutation=GCT->,position=4
#Sequence after mutation: ATGGAA.

Substitute GCT with AAATTT:

araC mutation mutation=GCT->AAATTT,position=4
#Sequence after mutation: ATGaaatttGAA.

residue_mutation¶

residue_mutation: Mutating a residue.

Options:

• mut Amino acid substitions. Multiple substitions can be separated by a semicolon (;)

Examples:

thiD residue_mutation mut=N5Q
#substitue N with Q at residue 5 in thiD.

Amino acid sequence before is MKRINALTIA, and after substitution it is MKRIQALTIA.

Deletions are denoted with a *:

thiD residue_mutation mut=N5*

Insertions are denoted as a an @ followed by an insertion number, lower-case suffix letter and then the inserted amino acid:

thiD residue_mutation mut=@5aA
#insert alanine in thiD after position 5.

thiD residue_mutation mut=@5aA;@5bA
#insert two alanines in thiD after position 5.

The symbol for stop codons is $:

thiD residue_mutation mut=N5$
#substitue N with a stop codon at
#residue 5 in thiD.