Part:BBa_K4146106
The complete expression box used for expressing caffeine.
The complete expression box used for expressing caffeine.
Our project is designed to help people improve their scalp microecology. After considering many comprehensive factors, we believe that E. coli Nissle 1917 is a safe, easy to operate and easy to absorb non-esterified fatty acids chassis. So all of these genes are in E. coli Nissle 1917.
From this picture, we could find many ways to synthesize caffeine. For example, a pathway of caffeine is a sequence consisting of xanthine → 3-methylxanthine → theobromine → caffeine. In three of the four reactions, the gene encoding N-methyltransferase was involved. And there is another way consisting of xanthosine→7-methylxanthosine→7-methylxanthine→theobromine→caffeine. Genes encoding N-methyltransferases involved in three of these four reactions have been isolated and the molecular structure of N-methyltransferases investigated. We finally chose the second way.
From this caffeine synthesis pathway, it’s clear that the gene of TcAncCS1 is derived from Cocobroma. Xanthine can be converted to 3-methylxanthine (3-mX) by TcAncCS1 from Theobroma. TcCS2 from C. sinensis has a higher propensity to recognize 3-mX and can convert 3-mX to theobromine. CS stands for caffeine synthase, while the TcAncCS1 ancestral gene is derived from Cocobroma, the duplication of TcAncCS1 at node O leads to TcAncCS2, but the kinetic parameters of TcAncCS2 and its progeny enzymes predict that experts will be favored because the 3 methyl Xanthine does not compete for active site binding. We introduced this plasmid gene to facilitate the synthesis of caffeine in engineered strains, thereby regulating the balance of scalp oil.
This is the plasmid diagram of the synthetic caffeine.
This is the picture of the theobromine’s standard curve. By analyzing its linear regression in the excel software, with R2=0.9995. It means that data can be well fitted to a straight line, with good linear regression. This standard curve is in line with the standard and can be used for the conversion of theobromine production.
Through the experiment, we found that the maximum yield of theobromine was 7.16mg/L.
This is the picture of the caffeine’s standard curve. By analyzing its linear regression in the excel software, with R2=0.9998. It means that data can be well fitted to a straight line, with good linear regression. This standard curve is in line with the standard and can be used for the conversion of caffeine production.
Through the experiment, we found that the maximum yield of caffeine was 1.83mg/L.
In the results, we can find that there are more theobromine than caffeine produced. Therefore, it was speculated that the lower methylase activity in the last step resulted in the accumulation of theobromine and affected the synthesis of caffeine.
Sequence and Features
- 10INCOMPATIBLE WITH RFC[10]Illegal PstI site found at 2479
Illegal PstI site found at 2602 - 12INCOMPATIBLE WITH RFC[12]Illegal PstI site found at 2479
Illegal PstI site found at 2602 - 21COMPATIBLE WITH RFC[21]
- 23INCOMPATIBLE WITH RFC[23]Illegal PstI site found at 2479
Illegal PstI site found at 2602 - 25INCOMPATIBLE WITH RFC[25]Illegal PstI site found at 2479
Illegal PstI site found at 2602
Illegal NgoMIV site found at 643
Illegal NgoMIV site found at 1741 - 1000COMPATIBLE WITH RFC[1000]
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