Part:BBa_K2671420

TetR-TetP-GroES

The gene coding for the chaperone GroES (homologue to HSP10 in eukaryotes) that is found in E.coli. It often works in conjunction to GroEL creating an environment well suited for the folding of proteins. NOTE: The sequence is codon optimized (E.coli) with IDT's tool. This sequence will not yield any results with BLASTn, but will hit GroES with BLASTx. The TetP also has one TetR binding site mutated (one base).

The gene coding for GroES is placed downstream from a tetracycline promoter. Further upstream the tetracycline repressor protein (TetR) is found, expressed by a constitutive promoter. The TetR protein has a termination sequence directly downstream from it, while the GroES gene has its transcription stop by the E.coli his operon termination sequence that is present in most pSB vectors, directly downstream of the insert.

Verification

We verified that our part was working as intended in two ways. After ligation it was sent for sequencing which gave the correct results, showing a successful assembly of the part into both pSB1C3 and pSB4A5, see attached files below. Secondly we verified a functional gene expression by SDS-PAGE analysis (see fig 1, 6).

Sequencing results for both the backbones used. File:T--Linkoping Sweden--GroES1C3.zip File:T--Linkoping Sweden--GroES4A5.zip

Figure 1. An induced BBa K2671420 in pSB1C3. A= Induction of GroES with tetracycline 200ng/ml, B= Leakage from adjacent wells, C= No induction of GroES, D= E.coli (BL21) without the GroES plasmid, E= BLUeye Prestained Protein ladder from Sigma Aldrich. GroES can be seen at 10 kDa. Induced bacteria containing the GroES plasmid has the highest Levels of GroES percent. The uninduced E. Coli containing GroES can be seen having some higher expression levels of GroES than the negative control cells. This is probably due to a slight leakage from the promoter.

Figure 6. An induced BBa K2671420 in pSB4A5. A= Induction of GroES with tetracycline 200ng/ml, B= Uninduced E.coli (BL21), C= leakage from adjacent wells, D= BLUeye Prestained Protein ladder from Sigma Aldrich. The arrow indicates the 10 kDa region where GroES is found.

Results

The results for our engineered system is shown below. Our biobrick is only called GroES in the graphs for simplicity. The substrate proteins have their names written out. We chose to include the GroE system and a combined GroES+GroE system to better characterize our part. GroE is a plasmid where both GroEL and GroES is expressed. The concentrations we used to induce all the different systems was: 200 ng/ml tetracycline for BBa_K2671420 (GroES), 0.5 mg/ml L-arabinose for the GroE plasmid and 0.5 mM IPTG for all the different substrates. All measurements were done in vivo in a 96-well plate. Excitation was done at 485 nm for all substrates and emission was measured at 520 nm. The chaperone plasmids, this biobrick and GroE was induced 30 minutes prior to the substrates, at an OD600 = 0.4 or close. After the 30 a minute headstart, the substrate proteins were induced and all combinations were placed in a 96-well plate. Absorbance at 600 nm was measured once at the start and at the end of the 16 hour experimental time. To note: all experiments was made with the part in pSB4A5 for co-express compatibility.

Conclusion and Future

Most substrates tested show an increase in intensity when used with our biobrick (true for all but α-synuclein-EGFP). To note also is that the kinetics of the substrates is slowed down by this part in some cases (see Figure 2 and 3) and is very clear in some cases when in concert with the GroE plasmid (see Figure 2, 4 and 5). Substrate proteins which tend to fold fast and misfold in the process might have a good use for this biobrick with or without the GroE system. Further research could compare our part against a confirmed holdase chaperone. It would also be interesting to investigate the binding mechanism between GroES-substrate, through perhaps crystallization or HSQC.

Figure 2. Results for mNG-Aß1-42. Top graphs showing three different systems of chaperones tested, GroES, GroE+GroES and only GroE. The bottom graphs show the normalized values from the top ones. The error bars illustrates the Standard Deviation.

Figure 3. Results for EGFP-Aß1-42. Top graphs showing three different systems of chaperones tested, GroES, GroE+GroES and only GroE. The bottom graphs show the normalized values from the top ones. The error bars illustrates the Standard Deviation.

Figure 4. Results for α-synuclein-EGFP. Top graphs showing three different systems of chaperones tested, GroES, GroE+GroES and only GroE. The bottom graphs show the normalized values from the top ones. The error bars illustrates the Standard Deviation.

Figure 5. Results for Tau0N4R-EGFP. Top graphs showing three different systems of chaperones tested, GroES, GroE+GroES and only GroE. The bottom graphs show the normalized values from the top ones. The error bars illustrates the Standard Deviation.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal NheI site found at 7
  Illegal NheI site found at 30
  
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]