Part:BBa_K4170054

rhaBAD operon

This plasmid contains the rhaBAD operon and rhaR-rhaS activators of E.coli flanked by BioBrick Preffix and Suffix regions into the pSB1C3 backbone. The rhaBAD operon encodes the L-rhamnose catabolic enzymes and the RhaS and RhaR regulatory proteins are encoded in the Escherichia colil-rhamnose gene cluster. The rhaBAD operon was amplified utilizing colony PCR amplification of DH5a competent E.coli cells.

Basic parts of the device

The part sample which is flanked at the beginning and the end with prefix and suffix respectively, is composed of the following basic parts assembled together in series and downstream of the prefix:

• Bacterial terminator (for LacI CDS) (located at the backbone): putative bacterial transcription terminator
• Biobrick Prefix sequence: BioBrick prefix for parts that do not start with "ATG"
• rhaR regulator: DNA-binding transcriptional activator for rhaSR, L-rhamnose binding
• rhaS regulator: DNA-binding transcriptional activator for rhaBAD and rhaT, L-rhamnose binding
• rhaB promoter: promoter of the E. coli rhaBAD operon, conferring tight induction with L-rhamnose and repression with D-glucose in the presence of RhaR and RhaS
• Biobrick Suffix sequence : universal suffix for all parts
• His operon terminator (downstream of Suffix) (located at the backbone): putative transcription terminator from the E. coli

Figure 1.Illustration of Cloned Rhamnose Repos BBa_K4170054 plasmid. Map was created with SnapGene.

Cloning strategy for device assemble with pSB1C3 backbone

For the final assembly of the PCR amplified genetic elements into pSB1C3 plasmid we followed the Golden Gate-based ‘SevaBrick Assembly’ method and the SEVA 3.1 platform. As for all our final composite parts, the initial steps of our cloning strategy constitute the PCR amplification of different genetic elements, followed by the efficient assembly of the PCR amplified genetic products into pSB1C3 backbone. The cloning process is described in detail below:

Step 1

• PCR amplification with Prham.Brk-F and Prha.BrkR primers from colony PCR of DH5a competent E.coli cells. This PCR produces the rhaBAD-rhaR-rhaS-colony part ready for Golden Gate assembly.

Figure 2: Colony PCR of E-coli DH5a using Prham.Brk-F and Prham.Brk-R primers from 4 different colonies in order to amplify the Rha sequence. 1 % agarose gel electrophoresis. Lanes 1-4: Rha (2091 bp) using Prham.Brk-F and Prham.Brk-R primers from different colonies. DNA ladder: 100bp DNA Marker NIPPON Genetics EUROPE.

After the completion of the colony PCR amplifications, 1 % agarose gel electrophoresis was conducted. As shown in figure 2 the rhaBAD operon (2091 bp)was slightly amplified from colonies 1 and 3 while amplification from colony 2 was unsuccessful. For our next experiments we used RhaBAD-rhaR-rhaS colony part amplified from colony 4 which was the most successfully amplified colony.

• PCR amplification with Ev and Pv standard primers from Basic SevaBrick Assembly [seva 3.1] using the Bba_J364007 part of the 2022 DNA distribution Kit. This PCR produced the pSB1C3 backbone linearized and ready for Golden Gate assembly.

Step 2

Golden Gate assembly of the PCR amplified rhaR-rhaS-colony part with the linearized pSB1C3 vector for the efficient construction of the rhamnose repository plasmid.

The products of the Golden Gate assembly underwent transformation into E.coli DH5a competent cells and then colony PCR was performed, using VR and VF2 primers (figure 3). For the colony PCR procedure, from the agar plate half amount of each colony was picked and diluted on 10 μl of dH20. The other half amount was picked for liquid overnight culture. Picking samples from different colonies and then evaluating the results on a 1% agarose gel electrophoresis, we concluded that the Golden Gate assembly was successful on colonies 3 and 4 (2345 bp).

Figure 3: Colony PCR of E-coli DH5a transformants using VR and VF2 primers, after Golden Gate assembly. 1 % agarose gel electrophoresis. Lines 1-4: Rhamnose repos using VR and VF2 primers (2345 bp) from different colonies. DNA ladder: 1kb plus DNA ladder NEB

Sequence and Features

Assembly Compatibility:

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

  Illegal NheI site found at 289
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 1318
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal AgeI site found at 1586
  
• 1000
  COMPATIBLE WITH RFC[1000]

Citations

[1] Egan, S. and Schleif, R., 1993. A Regulatory Cascade in the Induction of rhaBAD. Journal of Molecular Biology, 234(1), pp.87-98.

[2] Kolin, A., Jevtic, V., Swint-Kruse, L. and Egan, S., 2007. Linker Regions of the RhaS and RhaR Proteins. Journal of Bacteriology, 189(1), pp.269-271.

[2] Tobin, J. and Schleif, R., 1990. Purification and properties of RhaR, the positive regulator of the l-rhamnose operons of Escherichia coli. Journal of Molecular Biology, 211(1), pp.75-89.