Part:BBa_K1963003
A template for expression of sRNA fusions to the Serratia chiA hairpin
This is a multi-sequence system for small RNA (sRNA) production with co-expression of S. marcescens Hfq BBa_K1963001. The system has the proD strong promoter, followed by a chiA hairpin sequence from the 3'-end of the S. marcescens chiA gene encoding a chitinase, followed by the strong terminator sequence T1/TE. The use of an S. marcescens chiA hairpin/Hfq pairing should provide very high specificity in chosen chassis organisms. Phosphorylated primers should be used to insert the reverse/complement target sequence of your choice between the promoter and the hairpin sequence. The sRNA should be placed betweeen C-193 and G-194.
Usage and Biology
Some stuff here.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI.rc site found at 101
Functional Parameters
Follow this protocol and this BioBrick should provide you with a successful system:
1. Dilute template to approx. 20 ng/ul and use 1 ul of this in PCR reaction [NB. if you use too much template it will carry over and you will get template plasmid back after ligation and transformation]
2. Use Phusion DNA polymerase (it is high fidelity, fast and gives blunt ends)
3. Design forward and reverse primers. One will have extra sequence at the 5’ end corresponding to the reverse/complement of the target sequence. Order phosphorylated primers from your favourite suppliers.
4. Use Tm calculator to work out correct annealing temperatures to use [NB. REMEMBER TO ONLY CALCULATE Tm FOR THE BASES IN PRIMER THAT ANNEAL. You require approximately 20 bp that anneal perfectly to the plasmid template, thus your primer will be >44 bp in length (24 bp of overhanging sRNA targeting bases and 20 bp to anneal to plasmid)
5. DpnI treat your PCR product to get rid of template (1 hr at 37C should be fine)
6. Run everything out on agarose gel - be patient with this and resolve bands properly. Extract using a kit with a slight modification: put the dissolved agarose / binding buffer mixture through the column at least twice to make sure you catchall of the DNA. Then elute in smallest volume you can (30 ul maximum) - using warm, 50C, water will help increase elution efficiency.
7. Ligation: overnight at 16C works well, but room temperature for a couple of hours is also an option. Use all of the PCR product from the gel extraction (e.g. 30 ul DNA in a 35 ul ligation)
8. Transformation: the competent cells have to be good, so make sure you either buy some commercial ones or that you test the transformation efficiency of your own cells. Do a control transformation with the same concentration (20 ng) of the template plasmid used for PCR - this will tell you the max number of colonies you could expect to get from your ligation if everything was 100% efficient (it never is, you should get less). If using electrocompetent cells, use a maximum 3 ul of ligation mix to avoid arc-ing. If this leads to no transformants, dialyse your ligation mix to remove salts using Millipore filters floating on water in a petri dish for 1 hour and then use 6/7 ul.
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