The building of the construct to be tested occurs in four major steps: preparation of the 5’ UTR, plasmid preparation, ligation and transformation.
Preparation of the 5’ UTRs: Many of the 5’ UTRs that we ordered arrived as single-stranded oligonucleotides. These cannot be cloned on their own and hence require some processing which is as follows. The forward and the reverse strands are added to a PCR tube, passed through the thermocycler and then cooled gradually to room temperature. This yields double stranded UTRs which can now be cloned. Once double stranded, the 5’ UTRs are digested with enzymes XbaI and PstI so that they can be ligated into the plasmid vector.
Preparation of competent E. coli cells is done by inoculating the cells first in LB broth overnight, reinoculating some cells from the overnight culture into a new LB broth, centrifuging them, and resuspending the pellet containing the cells in cold calcium chloride solution.
Preparation of the Plasmid: We have used the plasmid pSB1C3 in our experiments. This plasmid is extracted from the cells and is then purified with several washings. The purified plasmid is then digested using SpeI and PstI enzymes for at least four hours at 37℃
Ligation: A 3:1 ratio of UTR:vector is used for the ligation. The digested plasmid and the digested P-UTR are added to a PCR or Eppendorf tube along with ligase buffer, T4 DNA ligase and water. The ligation mixture is incubated overnight at 16℃ using a thermomixer.
In this step, the 5’ UTR is ligated between the promoter and the RBS, and in case of P-UTR construct, it is ligated upstream of the RBS. We have used the part BBa_J23100 as our promoter, the part BBa0030 as our RBS and the monomeric RFP BBa_E1010 as our gene of interest. Our control has the setup Promoter---scar---RBS---scar---CDS, and has no 5’ UTR as such. This is because we are aiming to quantify the effect of the 5'UTR on gene expression.
We check for this using 1% Agarose Gels. This data is presented in our test section.
Transformation: The competent cells are thawed, mixed with the cloned plasmid and are then incubated on ice. Each transformation tube is heat-shocked by placing the tube into a 42°C water bath for 45 seconds. The tubes are then put back on ice. These cells are incubated in LB for 45 mins at 37°C, and are then plated on an agar plate containing chloramphenicol.
These cells are then incubated and fluorescence levels are mapped at regular intervals to characterize the effect of the 5’ UTRs.
The UTR parts are available on our Parts Page and the detailed protocol also has been uploaded on the Wiki.
On getting this experimental data, it is compared with the data generated by our dry lab using predictive software and models, and thus build our library of 5’ UTRs. This library is available as an open-source resource in the iGEM Parts Repository. All our constructed have been BioBricked by using BBrickIt. BBrickIt is a software that BioBricks any DNA sequence for you. According to the BioBrick standards, each BioBrick part must have a Prefix and Suffix. For a part to be compatible with BioBrick RFC[10] standards, it must not contain the following restriction sites, as these are unique to the prefix and suffix:
| Sequence | Type | Enzyme |
|---|---|---|
| gaattc | Illegal | EcoRIt |
| tctaga | Illegal | XbaI |
| actagt | Illegal | SpeI |
| ctgcag | Illegal | PstI |
| gcggccgc | Avoid | NotI |
If the sequence to be contains any of these sites, the program will detect them and they will be highlighted. The software will also suggest another sequence, with the illegal sequences replaced by new sequences which do not change the overall amino acid coding sequence. The most exciting feature of this application is that it takes into account the codon preferences for your chassis organisms. The frequency and efficiency of usage of codons in different organisms are different for producing the same amino acid. BBrickIt takes that into account and then suggests a new sequence that is optimized for the chassis organism.
BBrickIt v.2 builds upon the previous version of BBrickIt. This previous version of BBrickIt has been completely developed by our own ICT Mumbai iGEM Team of 2017. We have modified it this year. Users can now input an mRNA sequence in the input field and the software will convert it to the corresponding DNA sequence and return the BioBrick. It also comes with a “Copy BioBrick Sequence” button to enable users to copy the BioBrick sequence to their clipboard with the click of a button. Several bug fixes have also been made.
Steps to Use BBrickIt:
- Select your Organism/Chassis.
- Paste your sequence in the box at the top.
- Choose if your sequence is a coding sequence or not.
- Click BioBrick!
- If you wish to take the reverse complement of the sequence and then BioBrick it, click If your part contains any illegal restriction enzyme binding sites, two boxes will appear. The box on the left will display the highlighted restriction sites with a legend at the bottom. The box on the right will suggest the new, optimized sequence for the selected organism. If you wish to proceed with that sequence, copy and paste it into the top box and hit BioBrick.
As a result of using BBrickIt, all the parts uploaded on the Registry of Standard Biological Parts are BBF RFC 10 compliant.
While our project is currently limited to the E. coli system, we intend to broaden its scope to include other prokaryotic systems. We aim to characterize the effect of 5’ UTRs in eukaryotic cells, possibly even mammalian cells since these cells are usually employed to make several rDNA products, especially therapeutic substances. We also plan to insert our UTRs into expression vectors like the pET series of vectors to test how they perform in controlled gene expression systems. All this can be done conveniently and is well thought of.