C. Athale's page // IISER Pune

Bio122: Introduction to synthetic biology

[ver. 2019]

Here we aim to introduce concepts in synthetic biology and run you through a hands on exercise in virtual cloning of a gene for expression. The target gene is GFP.


Materials

  1. APE: a plasmid editor program
  2. pSB1A3 (Amp selection) http://parts.igem.org/Part:pSB1A3
  3. lacP promoter sequence (BBa_R0010) inside a pSB1C3 backbone http://parts.igem.org/Part:BBa_R0010
  4. rbs + GFPmut3 + ter1 + ter2 sequence allows us to include a ‘functional’ version of the sequence for translation (BBa_I13504) http://parts.igem.org/Part:BBa_I13504

Materials

All plasmids* can be downloaded from here.

Lab notebook: Questions to address

In your lab-notebooks note the following:
  1. The size of the 3 plasmids
  2. The schematic of the cloning strategy (i.e. draw the 3 plasmids you started with, the enzymes you digested them with virtually, the ligation reaction, planned transformation and selection.
  3. Sketch a representative figure of the DNA digest (in APE, choose "Enzyme Selection" ---> "Digest")
  4. Note the sequence of the transcriptional terminator in your construct.
  5. How the transcriptional terminator function? Pre- and Suff-ix DNA sequences

Protocol

Download the file: [pdf]

3A Assembly explained graphically

3A assembly- biobricks standard

Some notes from the presentation in lab (LHC303)

References


Last updated: 25-Feb-2019