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Re-Design of first IntI1 gBlock
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Three major changes happened to the gblock. We changed the code for the ribosome cut site which had one base wrong, we decided to put an Nhe1 restriction enzyme cut site between the inti1 gene and the promoters on the SamR construct because the low copy backbone we planned to move this biobrick to for validation had an Sac1 cut site in its backbone, and finally we totally overhauled the Gibson end for the junction between the pBAD promoter and the Inti1 gene because there was extra code on the SamR construct that we didn’t know about when designing this gblock the first time. These changed were performed by our supervisor Nick Coleman in consultation with Rokiah Alford.
The gibson assembly of SamR’s plasmid and the Inti1 gblock was successful as was checked in the following ways: junction PCR and sequencing, restriction digestion, biobrick PCR ad sequencing.
Plasmid Map of pUS203
To see this part on the iGEM Parts catalogue please click here. Sequence for this part are available in SnapGene (.dna file), FASTA (.fa file), GenBank Standard (.gb file) and Plain Text (.txt file) file format.
Design of IntI1 gBlock
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The code for the Inti1 gene was sourced from pUS2056 (theUSyd Coleman- Holmes lab had this LINK). We tried to get the part from the Paris Bettencourt team (from 2010 part BBa_K329014 http://parts.igem.org/cgi/partsdb/puttext.cgi) but this was not possible so we had to make it. It was modified by Tom Geddes, Rokiah Alford and Nick Coleman to include:
- - a different ribosome binding site,
- - to remove an illegal spe1 site near the end,
- - to include a Sac1 restriction enzyme site at the beginning before the inti1 gene so it could be excised independently of the promoter,
- - to remove a promoter pC that was coded in the opposite direction to the promoter we wanted to use and would have clashed with the controllable promotion of this part, and
- - to have Gibson ends that overlapped with the backbone we wanted to put it into which is described below.
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Our first attempt to Gibson Assemble these two – the Inti1 Gblock and SamR’s plasmid – failed because the gibson end that overlapped the araC-pBAD section was incorrect. There was extra code after the code for pBAD which we had not anticipated. This triggered the redesign of the gblock.
Construction
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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
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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
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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.
