Part of our project concerned an assembly that produced L-DOPA (levodopa or L-3,4-dihydroxyphenylalanine) from L-tyrosine in E. coli.
L-tyrosine can be converted to L-DOPA by the enzyme tyrosine hydroxylase. An equivalent of tyrosine hydroxylase in bacteria is 4-hydroxyphenylacetate 3-monooxygenase which is expressed by the genes hpaB and hpaC in E. coli. To simplify our assembly, we designed our construct to position hpaB and hpaC coding sequences in succession under the control of CP25 promoter which constitutively expresses downstream coding genes.
We constructed our production assembly by cloning a CP25-hpaBC-rpoc transcriptional unit into a vector containing a pMMB67EH ori and spectinomycin resistance marker. Our construct was assembled using golden gate cloning techniques. To do so, we first used golden gate to assemble all individual parts (CP25 promoter, hpaBC, rpoc terminator, and backbone) into entry vectors with the Type II restriction enzyme BsmBI. From there, the entry vectors were used to assemble a cassette plasmid with the Type II restriction enzyme BsaI. Cut sites for BsmBI and BsaI had been added previously to all parts by the Ellington Lab through the use of primers and PCR amplification.
We made a similar assembly using the same methods; in this assembly, however, we used PA3 promoter, another constitutively expressed promoter, in place of CP25 because we were afraid CP25 was too strong of a promoter.
Here is a brief overview of each main constituent of the hpaBC transcriptional unit:
CP25 promoter strong promoter; initiates transcription of hpaBC; interchanged with PA3 promoter.5 promoter. PA3 promoter (BBa_K2458000): moderately-strong promoter; initiates transcription of hpaBC; interchanged with CP25 promoter.
hpaBC (BBa_K2458001): gene expressing the enzyme hpaBC (4-hydroxyphenylacetate 3-monooxygenase).
rpoc terminator (BBa_K2458002): terminator; terminates transcription of hpaBC.
The second half of our project revolved around an assembly that sensed the production of L-DOPA.
In our sensing assembly, we employed the transcriptional factor pp2551, a member of the lysr family of transcriptional factors,
Our sensing construct consisted of two primary transcriptional units: one with the pp2551 coding gene and one with the Venus fluorescent protein coding gene. We constructed this assembly using golden gate, gibson, and traditional cloning techniques.
We had some trouble cloning our sensing assembly, and as a result, we transitioned from using golden gate assembly methods to gibson assembly methods to traditional cloning techniques. Initially, we attempted to clone our assembly using gol
Here is a brief overview of the transcriptional units and their functions.
lacI Transcriptional Unit -
pp2551 Transcriptional Unit - expresses transcriptional factor pp2551 in the presence of DOPA. CP25 promoter strong promoter; initiates transcription of gene expressing pp2551. pp2551 (BBa_K2458005): gene expressing the transcriptional factor pp2551. ilvGEDA terminator (BBa_K2458006): terminator; terminates transcription of gene expressing pp2551 transcriptional factor.
Venus Transcriptional Unit - ppDDC promoter (BBa_K2458007): promoter activated by pp2551 transcriptional factor; when pp2551 binds to ppDDC, transcription of coding gene increased; promoter initiating transcription of Venus fluorescent protein coding gene. Venus fluorescent protein coding gene for Venus fluorescent protein. rpoc terminator (BBa_K2458001): terminator; terminates transciption of Venus fluorescent protein coding gene.