From 2013.igem.org

Uppsala iGEM 2013

Results and achievement

Achievements

Successfully managed to clone and sequence completely new biobricks. Read more

Improved already existing biobricks. Read more

Successful characterization of several biobricks. Read more

Helped another iGEM team. Read more

Executed several successful human practice projects Read more.

Created a new standard backbone for Lactobacillus. Read more

Useful mathematical modeling of metabolic pathways. Read more

Completed our chromoprotein collection and displayed 8 new chromoproteins

Results

p-Coumaric acid

We managed to clone out and biobrick tyrosine ammonia lyase and verify the biobrick by sequencing. Also we did succeful characterization on this part, showing that it works as expected. We managed to express our enzyme and detect it in a western blot, and also detect our metabolite in both spectrophotometry and hplc. The biobrick was characterized in E. coli d5 alpha and E. coli nissle. For detailed information about the characterization methods, see the protocol section. To read more about p-coumaric acid, click here

1. Positive control
2. TAL with Cp8 promoter
3. TAL with J23110 promoter
4. Negative control

Figure 1:SDS-page and western blot. Expression of Tyrosine ammonia lyase with constitutive promoters. The negative control is empty, showing that there is no natural protein in E. coli with the same attributes.

Figure 2:Absorbance spectra of extracts collected from bacterial cultures. Samples were collected 21 h and 48 h after 30 °C incubation. The negative control is an extract from a strain with no TAL gene on transformed plasmid. The positive control is an extract a culture of the same strain as the negative control but with added p-coumaric acid to a concentration of 500 µM before extraction. p-Coumaric acid absorbance spectra has two peaks. The one around 305 nm is preferable to detect because of background from bacteria. (a) Spectra from the strain with TAL CDS with promoter J23110.(b) Spectra from the strain with TAL CDS with promoter CP8. (c) Spectra from the strain with TAL CDS with promoter J23101.

	Figure 3: Graph showing the HPLC result of a sample prepared from E. coli expressing tyrosine ammonia lyase. Reverse phase HPLC with a C18 matrix was used. The peak for p-coumaric acid can be seen ~10 min, as shown by the standard sample below.
	Figure 4: Graph showing the HPLC result of a sample standard with p-coumaric acid.
	Figure 5: E. coli culture injected to the hplc without our biobrick tyrosine ammonia lyase. Here we can see that there is originally no peak at 10 minutes.

Resveratrol

Although we managed to clone out and sequence verify the genes for resveratrol production, we have had some problems in the characterization. The results are unclear, and we did not have time for further investigations.For detailed information about the characterization methods, see the protocol section and to read more about resveratrol, click here

Figure 6: Number 2 shows a very weak band of our protein at around 43 kDA.
Positive control -> 1, Stilbene synthase -> 2

4Cl-STS translational fusion expressed in E. coli

Figure 7: E. coli supposed to produce resveratrol. As we can see, we got very low absorbance peaks at ~30 min, ~33 min and ~36 min.

Resveratrol standard

Figure 8: Resveratrol standard, peaks around 33-34 min.

Resveratrol standard scaled

Figure 9: Resveratrol standard that is scaled down to correspond to the absorbations of our E. coli supposed to produce the corresponding metabolite. The peaks are at around 33-34min.

Lysed bacterial culture without plasmid of assembly

Figure 10: E. coli culture injected to the hplc without our biobrick tyrosine ammonia lyase. Here we can see that there is originally no peaks around 30-35 minutes.

Shuttle vector

We constructed two shuttle vectors able to replicate and provide resistance in both Lactobacillus and E. coli. We have shown that they can both be used in E. coli for assembly and subcloning as well as transforming them to Lactobacillus. To read more about the shuttle vector, click here

Figure 11: Here you can see our shuttle vector BBa_K1033206 with the insert BBa_K1033282 in E. coli, after growing for 24 hours. This showed that we can subclone a new construct to our shuttle vector and that it works in E. coli.

Figure 12: Here we can see a transformation of BBa_K1033207 to Lactobacillus reuteri 100-23. The plate on the left is the result of a transformation with our shuttle vector and the plate on the right is without the shuttle vector. Controls were also done on antibiotic free plates yielding hundreds of colonies.

Promoters

We have provided a constitutive promoter collection with 6 promoters with different strength measured by a fluorescence-activated cellsorting machine (FACS) in RPU, a unit based on the strength of the promoter J23101 in standard parts. These promoters works in both gram-negative bacterium E. coli and gram-positive bacterium Lactobacillus and are therefore also believed to be universal for all prokaryotic organisms. To read more about promoters, click here

Figur 13: The CP promoters strength relative to J23101 in E. coli.

Competition test

We grew our E. coli Nissle containing the TAL biobrick together with wild type Nissle (mixed 1:1) in antiobiotic free LB medium. The culture was maintained by a thousandfold dilution in new medium every day, and letting it grow again overnight. To be able to track the fraction of each population over time, a dilution of the culture was plated on both selective, and non selective plates. The amount of colonies on the non selective plates gives us the total amount of cells, while the amount of colonies on the selective plates gives us the number of cells carrying the biobrick plasmid. The fraction of cells carrying the plasmid declined rapidly, and already after 5 days (approximately 50 generations of growth), we saw no trace of our E. coli Nissle with TAL on the selective plates, while the wild type still grew on the non selective plates. This clearly shows that our genetically engineered probiotic have a hard time competing against the wild type, and if released into the environment, they would most likely be outcompeted by other bacteria within days. To read more about the competition test, click here

Competition experiments between TAL and wild type

Figure 13: Competition experiments between E. coli Nissle carrying the TAL plasmid and wild type Nissle.

Team:Uppsala/results