Team:USyd-Australia/Parts

Plasmid Map of pUS203

Design of IntI1 gBlock

Re-Design of first IntI1 gBlock

Construction

The IntI1 gBlock was inserted into the SamR construct using Gibson Asssembly. The ends of the gBlock in the redesign of the gBlock were compatible with the ends of the SamR construct and after running the Gibson Assembly reaction we used a variety of methods to prove that our gBlock had inserted correctly (See Below Proof section)

Proof

Junction primer PCR: The colonies from the Gibson Assembly reaction of the SamR plasmid and the Inti1 gblock that grew of the LB+ Cm25 plates were screened using two sets of junction primers. One set was for the junction between the promoter and the gblock, the second for the junction of the gblock and the PSB1C3 backbone.

Junction primers for the junction of promoter and gblock were iGEM1413 and iGEM1414. These yielded a fragment size of 377bp which was found in 9 out 12 of colonies sampled and patch plated. This result was far more than we anticipated because the selection method was only or the backbone’s resistence not the insert which was only the modified integrase gene. Samples from the patch plate of these positive colonies were subjected to a second junction primer screening. The junction primers for this were iGEM1416 and iGEM25 (last year’s USyd iGEM team designed this primer to amplify the whole biobrick from the suffix end and we re-purposed it here). These yielded a fragment size of 432bp in 8 of the expected 9 colonies that were positive for the 1st junction screen. From the colonies positive for both junction screens 3 were plasmid prep’d (colonies 3, 9, and 33).



Junction primer sequencing:


The products of both junction PCRs of these three colonies were sequenced and all sequences (the forward and reverse directions for each of the junctions i.e. 4 sequences per colony) corresponded to that predicted for the junctions using the code of the SamR plasmid and the gblock we designed.



Plasmid Prep and check with restriction digestion:


These three plasmid preps were checked by restriction digestion using combinations of an enzyme from the suffix (Spe1 and Pst1) and prefix (EcoR1), as well as Sac1 which has no cut sites and nhe1, an enzyme that cut only once at a site we introduced into the gblock for this purpose and to allow others to move the inti1 gene independently of the rest of the biobrick. The results were as predicted using snap gene for all except the Sac1 digest. We were satisfied with these results despite the Sac1 error because the other fragments were as expected and most importantly because Nhe1 cut, which could only have occurred if the gblock had been incorporated into the plasmid. These results supported the junction primer results. The enzyme in the Sac1 digest of pUS203 was heat killed and this digested plasmid prep was used for the PCR of the biobrick. This showed that the unexpected action of the Sac1 seen in the gel for the digest of this plasmid luckily didn’t reflect an error in the biobrick code.



Biobrick PCR and sequencing:


The final check for this plasmid was to PCR the entire biobrick fragment using iGEM16 and iGEM25 (last year’s USyd team’s primers for the biobrick). This yelided a fragment size of 2676bp in 6 out of 8 PCR reactions of heat killed Sac1 restriction digested pUS203. This PCR product was sequenced using the iGEM16 and iGEM25.

Validation

We made several attempts to validate this biobrick using 2 low copy backbones and finally just as it was. The low copyback backbones were desired because Inti1 is toxic to the cells at high concentrations.

The first attempt was to put it into a low copy backbone we’d made pUS202. The iGEM provided backbone was not accetpable as it used an antibiotic resistance gene that did not fit with our validation design. This backbone tested as high copy and therefore did not serve our needs. The second attempt was to move our biobrick by restriction digestion, phorphorylation and blunt end ligation into pSB6A1 which had had its rfp digested out and was de-phosphorylated. This was not succesful and time constraints pushed us to option three which was to use the biobrick in the high copy PSB1C3 that we submitted to the library.

The validation design uses three plasmids to form a working integron. There is an integrase plasmid (pUS42), and atti site plasmid (pUS44) and a plasmid with a cassette (i.e. attc sites) (pUS25 and pUS23), all of which were present in the lab we worked in and we proved work together to express the resistance of the cassette which had not been expressed before the combination of these plasmids in one call. All of these plasmids were in the JM109 strain of E. coli and all validation work was performed in this strain because top10 – the strain we used for making the plasmids has streptomycin resistance in its chromosome and would have given false positives fro the presence of plasmids.

Once these parts were proven to work together our integrase (in whichever form) was to besubstituted for pUS42 which had intigrase expressed constitutivly to prove it worked (i.e. made the genes of the cassette express) and that this expression was controllable. If integrase was proven we hoped to substitute in the casette we made (aaedB and Gm) in for pUS25/23 and have the system opperating with 2 pats we’d made and only one from the lab.