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From 2014.igem.org

pUS203

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.

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Design of IntI1 gBlock

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:
1. - A different ribosome binding site,
2. - To remove an illegal Spe1 site near the end,
3. - To include a Sac1 restriction enzyme site at the beginning before the IntI1 gene so it could be excised independently of the promoter,
4. - 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
5. - To have Gibson ends that overlapped with the backbone we wanted to put it into which is described below.
6. 
   
This backbone, made by honours student Sam Ross, is PSB1C3 with araC-pBAD (which was sourced from BBa_K731201 a well characterized part) in between the suffix and prefix and will be called SamR’s plasmid throughout our documents. The code for the BioBrick section of this plasmid was used to predict the sequence for junction PCR sequences and our BioBrick’s sequence.
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.

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Re-Design of first IntI1 gBlock

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.

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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)

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Proof

These changed were performed by our supervisor Nick Coleman in consultation with Rokiah Alford. The redesigned gBlock was able to be successfully Gibsoned into the SamR backbone. To verify the presence of the insert we used junction primers which would amplify over both ends of the insert and could only amplify if the gBlock had been integrated into the SamR construct. Junction primer PCR: Junction primers for the junction of promoter and gblock were iGEM1413 and iGEM1414 and were expected to give a fragment size of 377 base pairs.
9 out of the 12 colonies screened gave a positive result and these were further screened with a secon
d set of junction primers on the other side of the gBlock. The junction primers for this were iGEM1416 and iGEM25 and were expected to yielad a fragment size of 432bp.

Again 9 of the 12 colonies were positive here. The PCR product was sequenced and corresponded with the expected sequence for the plasmid. 3 of the 8 colonies were selected to be grown up and Plasmid Prepped to remove the plasmid and allow restriction digest and sequencing of the plasmid for further verification. Restriction Digest The pUS203 plasmid was further confirmed with restriction digestion. Spe1, Pst1 and EcoR1 are in the prefix and suffix and should cut. An Nhe1 restriction site was added into the gBlock code and so a cut with this enzyme would indicate the gBlock had been inserted. Sac1 should not cut and should be identical to the uncut control lane.

The digest results were as expected using SnapGene’s Virtual Digest tool. 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. The Sac1 sample was heat-treated to inactive the Sac1 enzyme and used was used as a template to PCR the BioBrick. The results were still as expected which 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 and sequencing the product. Back to top



Validation

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.
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