Part:BBa_K2560272

tfoX of V. cholerae

As an extension of our basic part tfoX we created a full transcriptional unit enabling you to perform natural transformation in V. natriegens. First we started assembling a full transcriptional unit using a constitutive promoter. Therefore, we assembled tfoX together with the constitutive promoters J23100 or J23110 into a transcriptional unit by golden gate assembly. After transformation of this reactions into E. coli we were able to get colonies that seemed to be positive for the Lvl1 plasmids encoding tfoX because they showed the right size of fragment after plasmid preparation and test digestion, whenever we sent them for sequencing, some basepairs of the tfox gene were always missing (Figure 1).

https://static.igem.org/mediawiki/2018/3/3e/T--Marburg--tfoxsequencing.svg

Figure 1:Alignment of sequencing results of our first constructed Lvl1 tfoX plasmids showing missing basepairs at places at which black fragments are missing.

We suggest that these problems could be caused because Tfox could somehow ne toxic to E. coli cells. At this point of our project we realized how important it is to have well characterized, tight, inducible promoters for the usage in V. natriegens. In addition to constitutive promoters, the Marburg Collection contains the inducible promoters, pTet and pTrc. From our measurements of the promoter characterization, we learned that the pTet promoter is the tightest one showing the lowest value of Luminescence without induction (Figure 2).

Figure 2: Relative promoter strength of Anderson promoters Data were normalized over the strongest construct J23100. Error bars represent the standard deviation of the measurements of three independent experiments

The pTet promoter can be induced by the tetracycline derivative anhydrotetracycline (ATc). ATc is much less cytotoxic than IPTG but still capable of binding and altering the structure of the repressor TetR, leading to release of the promoter and enabling transcription. Alignment of sequencing results of our first constructed Lvl1 tfoX plasmids showing missing basepairs. Using this suitable promoter for V. natriegens we successfully assembled a plasmid containing a full transcriptional unit with a pTet promoter and did not had any problems during the cloning process. By Test Digestion and sequencing we could prove, that now the sequence of Tfox was correct (Figure 3 and 4).
https://static.igem.org/mediawiki/2018/6/64/T--Marburg--Testdigesttfoxlevel1.svg
Figure 3:1 %- Agarose gel of the test digest. 2-logDNA ladder is used as marker on agarose gels.


Figure 4:Alignment of sequencing results of our final constructed Lvl1 tfoX plasmid showing the correct sequence.

We were able to perform natural transformation using this plasmid and could demonstrate, that GFP which is encoded on pYTK is expressed by V. natriegens.
https://static.igem.org/mediawiki/2018/e/ed/T--Marburg--pYTKtransformation.svg
Figure 5:Plate showing a successful transformation of pYTK using natural transformation. Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI site found at 1
  Illegal BsaI site found at 825
  Illegal BsaI site found at 1512
  Illegal BsaI.rc site found at 799
  Illegal BsaI.rc site found at 863
  Illegal BsaI.rc site found at 1659
  Illegal SapI.rc site found at 1048