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Team:USyd-Australia/pUS203
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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) 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 the pC promoter 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.
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.
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.
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To validate our araC-pBAD/IntI1 BioBrick it would need to be present in a low copy plasmid in order to reduce the amount produced as it is potentially toxic to the cell at high concentrations. Our intial plan was to insert the BioBrick into pUS202 but it turned out not to be low-copy after validation. The back up option was to ligate the BioBrick into pSB6A1. The initial attempt was not successful and due to time constraints we decided to try and test the integron construct in the pSB1C3 it was currently in even with the potential toxicity.
To test for the proper expression of our integron gene in the BioBrick we wanted to have an AttI site and integrase protein present in E. coli. This was so that upon transformation with a cassette (which has an AttC site) a co-integrate would quickly form with the AttI site and before the cassettes could be lost out of the cell. Upon integration of the cassette the Gentomycin resistance (GmR) present on the cassette is expressed allowing us to select for the correct cells on Gentomycin plates. The negative control had no arabinose induction and so the cassette should not integrate and the cells should not be Gm resistant.
The initial experiments did not yield positive results and due to time constraints we have not been able to troubleshoot the experiment.
