Team:Stuttgart/Engineering

Home
Team Members Attributions Collaborations
Contribution Project Description Safety
Engineering Success Parts Experiments Protocols Notebook Results
Human practice Education
Judging Form

Engineering Success Engineering Success

Idea

The Plasmid should contain the Dsup sequence and a His-tag. The addition of a tag could disturb the interaction of Dsup with the DNA. As a result the His-tag was inserted at the C-Terminus and the N-Terminus. We worked with a optimized sequence for E.coli to reduce the risks of unwanted influences.
For the HeLa plasmid, we decided to construct only one fragment with a His-tag on the N-Terminus.

A vector with dsup and His-Tag and without the addition of the His-tag was designed
The dsup sequence can be found on database (https://www.ncbi.nlm.nih.gov/protein/BAV59442.1 retrieved on 15.06.21) and codon optimized for the work with Escherichia Coli and HeLa-Cells by using the "Codon Optimization Tool" from IDT. According to the rules of iGEM, the fragments should not contain any restriction sites that are part of the BioBricks. This has already been taken into account in the cause of the optimization.
Snapgene was used to insert the gene into a suitable plasmid and determine primers for the Gibson assembly. Four dsup constructs were designed, three for E. coli (N-His; C-His and wt) and one for HeLa. The backbones pBAD18 (E. coli) and pcDNA3.1 (HeLa) were used. The part BBa_K3780011 (wild-type Dsup) contains the dsup, the prefix BioBrick and the suffix BioBrick as well as the arabinose promoter and the promoter region. BBa_K3780017 (N-terminal His-tag Dsup) contains the same parts but the His-tag is located behind the ATG-Codon. In BBa_K3780013 (C-terminal His-tag Dsup) the His-tag can be found in front of the stop codon. The dsup that was optimized for HeLa has a His-tag in the N-terminus (BBa_K3780019).
All sequences for the fragments and primers can be found in the Wet-Lab section.

Fig. 1: Vector map of empty vector pBAD18 shown by Snapgene.

Fig. 1: Vector map of empty vector pBAD18 shown by Snapgene.

Fig. 2: Vector map of wildtype dsup with both BioBricks optimized for E. coli, shown by Snapgene.

Fig. 2: Vector map of wildtype dsup with both BioBricks optimized for E. coli, shown by Snapgene.

Fig. 3: Vector map of N-terminal dsup with both BioBricks and His tag in N-terminus optimized for E. coli, shown by Snapgene.

Fig. 3: Vector map of N-terminal dsup with both BioBricks and His tag in N-terminus optimized for E. coli, shown by Snapgene.

Fig. 4: Vector map of C-terminal dsup with both BioBricks and His tag in C-terminus optimized for E. coli, shown by Snapgene.

Fig. 4: Vector map of C-terminal dsup with both BioBricks and His tag in C-terminus optimized for E. coli, shown by Snapgene.

Fig. 5: Vector map of empty vector pcDNA3.1(+) shown by Snapgene.

Fig. 5: Vector map of empty vector pcDNA3.1(+) shown by Snapgene.

Fig. 6: Vector map of N-terminal dsup with both BioBricks and His tag in N-terminus optimized for HeLa, shown by Snapgene.

Fig. 6: Vector map of N-terminal dsup with both BioBricks and His tag in N-terminus optimized for HeLa, shown by Snapgene.

Cloning and Expression

After the fragments arrived, we started the cloning process. Our E. coli fragments contain an arabinose promoter. The arabinose promoter is a tight promoter, which means that in the absence of arabinose, dsup cannot be expressed, so we can control the expression. We used the Gibson assembly cloning method because it is easier and cheaper than the other methods. The first transformation took places for all fragments in DH5α, because this E. coli strain is very suitable for transformation due to its high transformation efficiency. The second transformation was performed only for the E. coli fragments in BW25113. E. coli strain BW25113 is arabinose-deficient, which is advantageous for our case, as we induce with arabinose and can thus be sure that the E. coli cells do not consume arabinose. Since we did not know at which temperature dsup folds best, we performed the expression at 3 different temperatures: 37°C, 30°C and room temperature. Unfortunately, we did not have enough time to see at which temperature the protein folds best, so we decided to continue with the protein that had been expressed at 30°C.

Experiments

In order to confirm that Dsup protects cells intracellularly and extracellularly from UV damage, two projects were devised. For the intracellular experiment, all genotypes of Escherichia Coli are used. Overnight cultures were induced with arabinose and some genotypes were not induced with arabinose and incubated overnight at 30°C. After incubation, the optical spectrum of the cells was measured. After incubation, the optical density was measured at 600 nm and the right dilution was selected to achieve 100 colony-forming units on the plate. The plates were irradiated at different energies per area and incubated overnight at 37°C. The growth of the colonies on the plates were documented the next day.
To use Dsup to protect surfaces, the protein needs to work extracellulary. The protein was overexpressed and isolated from E.coli cells. The purified and concentrated protein was added to RPMI media and used during the experiment to cultivate the HeLa-cells. A live-cell imaging experiment was performed to find out if there are protective properties due to the Dsup. To compare the results, the experiment was performed with and without adding the Dsup as protective agent.