Team:Acton-BoxboroughRHS/Research

Pre-Research

E. Coli vs. Yeast

E.coli can live in a higher range of pH and should be easier to engineer, but may be less productive. Yeast will most likely be more be more productive in protein production in the long run but is otherwise inferior to the use of E. coli, so our decision is to use the latter. Also, we used a k12 strain of E. coli from Carolina Labs and a highly-competent strain of E. coli from New England BioLabs.

We need an open reading frame (orf) a ribosome binding site (rbs), a promoter, at least 3-5 enzymes:(pepsin, salivary amylase, trypsin, chymotripsin, and pancreatic amylase), and a terminator, to create a fully functional plasmid to digest the coffee beans.

We planned on using the amylase orf that already existed in our ordered tray, in part to treat the coffee beans for breaking down complex carbohydrates to simple sugars. This will make the resulting coffee less bitter and more sweet. The part BBa_K523006 codes for amylase and also includes it's own secretion mechanism, which enables the displaying of amylase on the cell’s membrane.

A promoter is a DNA sequence that tends to recruit transcriptional machinery and lead to transcription of the downstream DNA sequence. The part we anticipated to use as a promoter was BBa_J23100; however, our Amylase ORF already comes with its own LacZ promoter, and ribosome binding site.

A ribosome binding site (RBS) is an RNA sequence found in mRNA to which ribosomes can bind and initiate translation in bacteria. The part we anticipated was BBa_B0030. This part is considered a strong RBS in terms of initiating transcription. It has many 'relatives', such as BBa_B0031, BBa_B0032, BBa_B0033 and BBa_B0034.

Protein domains are portions of proteins cloned in frame with other proteins domains to make up a protein coding sequence. Some protein domains might change the protein's location, alter its degradation rate, target the protein for cleavage, or enable it to be readily purified. The open reading frame we actually ended up using in the main lab and the high efficiency lab was almost solely amylase, which as described, has its own transcription and secretion mechanisms.

The Terminator is the 'last' region of the plasmid, where RNA polymerase stops transcription. There are two main types of terminators in prokaryotes, rho-dependent terminators, and rho-independent terminators. Rho-dependent termination bases its function on the Rho protein, which causes the RNA polymerase to fall disassociate form the plasmid.

Rho-independent termination does not use Rho to disassociate the RNA polymerase, but instead stops by the DNA sequence, here's how it works. The DNA sequence contains a two regions that are rich in cytosine and guanine. When this is transcribed, those regions on the RNA form hydrogen bonds to itself: effectively forming a 'loop' which disassociates the DNA polymerase from the DNA.

Within the category of rho-independent terminators, are terminators that stop transcription on the forward strand in forward direction (forward terminators), on both strands and directions (bi-directional terminators), and on the reverse strand and direction only (reverse terminators).

One specific part that seems promising is the rho-independent, forward terminator BBa_B1006, which has a 99 per cent success rate of terminating the transcription, the highest efficiency of all the terminators.

A plasmid is a circular, double-stranded DNA molecules typically containing a few thousand base pairs that replicate within the cell independently of the chromosomal DNA. A plasmid backbone is defined as the plasmid sequence beginning with the BioBrick suffix, including the replication origin and antibiotic resistance marker, and ending with the BioBrick prefix.

This gene will help digest protein in the coffee bean. We needed to find a pepsin gene more similar to the Paradoxurus hermaphroditus (Asian Palm Civet), because there are many different variations. Any secretion most likely means lysis with acid: we do not have the acid proof backbone in our plate. Although we planned on getting this pepsin part synthesized using the template from the existing planned pepsinogen part BBa_M143, analysis of the sequence revealed multiple errors. The part claims to be optimized for production in E. coli; however, it includes the human signal peptide which would not work if synthesized in E. coli. By the time we realized this, it was too late to plan and synthesize a new part. So we decided to just plan the part, submit it, and get it synthesized in the future.

• Jumhawan, Udi et al, Selection of Discriminant Markers for Authentication of Asian Palm Civet Coffee(Kopi Luwak): A Metabolomics Approach
• NCBI Taxonomy Browser
• Martinez, Federico Luis, Quality Enhancement of Coffee with Acid and Enzyme Treatment
• Quality Enhancement of Coffee with Acid and Enzyme Treatment: Patent Application
• Marcone, Massimo F, Composition and properties of Indonesian palm civet coffee(Kopi Luwak) and Ethiopian Civet Coffee

End of Page