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Phosphofructokinase 1

This part contains a 6-phosphofructoskinase from Lactobacillus Johnsonii ATCC33200 that metabolizes fructose-6-phosphate to fructose-1,6-bisphosphate. This part is RFC 10 compatible. The part's codon usage is optimized for expression in lactobacilli. The codon usage of this part was optimized to balance DNA synthesis and expression in L. Johnsonii. Therefore, longterm and shortterm repeats had to be modified and biobrick restriction sites were removed. The part was modified for RFC10 at the prefix and suffix of the part.

BBa_K1882005

LDH-Promotor Phosphofructokinase

This part encodes BBa_K1882002 and BBa_K1882000 resulting in a L. johnsonii ATCC33200 optimized phosphofructokinase under the control of a constitutive LDH-promotor. This leads to overexpression of the phosphofructokinase in L. johnsonii ATCC33200 when transferring this part into a lactobacillus expression vector.

BBa_K1882001

LDH-Promotor

This part contains a the promoter of the L. johnsonii ATCC33200 lactate dehydrogenase, a constitutively expressed protein. This part is RFC10 compatible

BBa_K1882002

Fructose Transporter

This part encodes a carbohydrate transporter from L. johnsonii ATCC 33200 that imports fructose into the target cell. In this process, fructose is phosphorylated by an N-phosphohistidine residue to yield fructose-6-phosphate and histidine. The protein is part of the ABC transporter superfamily. This part ist RFC 10 compatible. The part's codon usage is optimized for expression in lactobacilli. The part was optimized to balance DNA synthesis by IDT and expression in L. johnsonii. Therefore longterm and shortterm repeats had to modified and biobrick restriction sites were removed.The part was modified for RFC10 at the prefix and suffix of the part.

BBa_K1882006

LDH-Promotor

This part contains a the promoter of the L. johnsonii ATCC33200 lactate dehydrogenase, a constitutively expressed protein. This part is RFC10 compatible

BBa_K1882007

Ketohexokinase

This part encodes a ketohexokinase (ATP:D-fructose 1-phosphotransferase) from Rattus norvegicus optimized for expression in Saccharomyces cerevisiae. The ketohexokinase converts fructose to fructose-1-phosphate using ATP to perform the phosphorylation. This protein is not common in yeast and functions as toxin of the cell by trapping phosphate and therefore ATP in fructose-1-phosphate, not being able to be further metabolized. This result stop of growth of transformed yeast in fructose containing media (Donaldson et al., 1993). This part is RFC25 and RFC10 compatible.

BBa_K1882000

ApaI-constPromotor

This part encodes for BBa_J23102 with an ApaI restriction site upstream of it. Combination with BBa_K1882010 enables this part to clone a construct in a RFC compatible plasmid.

BBa_K1882009

Terminator-Bam-Hind

This part encodes for the rrnB T1 Terminator (BBa_B0010) followed by a T7 Terminator (BBa_B0012) and BamHI and HindIII restriction sites. Combination with BBa_K1882009 enables this part to clone a construct in an RFC compatible plasmid.

BBa_K1882010

RBS-PezT

This part encodes for a codon optimized version of UDP-N-acetylglucosamine kinase (PezT toxin). Upon expression the toxin produces UDP-N-acetyl-alpha-D-glucosamine 3'-phosphate which inhibits MurA and therefore cell wall synthesis in E. coli as well as L. johnsonii. Besides the sequence for the PezT toxin this part contains an upstream located ribosome binding site and could therefore be cloned direct behind any promoter of choice.

BBa_K1882011

ZF ZFN-L

This part contains the zinc finger ZF ZFN-L, see also Lombardo et al., 2007. The part is compatible with RFC10.

BBa_K1882012

ZF K230R

This part contains the zinc finger ZFK230R, see also Lee et al., 2010. The part is compatible with RFC10.

BBa_K1882013

ZF-C7C7

This part contains the zinc finger ZF-C7C7, see also Liu et al., 1997. The part is compatible with RFC10.

BBa_K1882014

Kill 1

This part is a composite part of the following components: Apa-pRha + RBS-PezT + Terminator-Bam-Hind. It is RFC10 compatible, and can be used for negative selection.

BBa_K1882015