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

Lab notebook: Questions to address

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?

Protocol

3A Assembly explained graphically

Some notes from the presentation in lab (LHC303)

References

• BioBuilder by Nathalie Kuldell (available in the library @iiserpune): A synthetic biology lab book
• International Genetically Engineered Machines (iGEM): the not for profit foundation that conducts an annual contest of synthetic biology using OpenSource DNA and draws schools, colleges and universities from around the world
• iGEM2015 - the IISER Pune team Mycobacterium revelio
• Thermo Fisher- guide to Traditional Cloning
• Abdallat et al. (2014) Overexpression of the transcription factor HvSNAC1 improves drought tolerance in barley (Hordeum vulgare L.) Molec. Breeding. 33:401