Team:SydneyUni Australia/Notebook

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Overview of What We Did

Week 1

We performed a plasmid extraction of ToMo from E.coli (strain?). We then digested pBBR1-MCS2 using XbalI followed by gel of these samples including a marker and undigested pBBR. (link protocol- for plasmid prep, digest and gel)

Week 2

We used E.coli Epi300 to transform our two plasmids (pBBR and pBS-ToMO). Both contain Kanamycin resistant genes, allowing us to select for successfully transformed cells by growing them on LB-Km. E.coli (pSB-ToMO) was not growing well so transferred them to broth for further incubation. E.coli (pBBR) and E.coli (Psb-ToMO) were transferred and incubated in phosphate buffer solutions containing either 1mM DCA or no DCA overnight.

Week 3

We used the Gas Chromotrographer (GC) [hyperlink to chloride assay] to look at our two DCA treatments of E.coli. Both had similar results, which is not what we were hoping for. Two possible explaination for this may be that either ToMO was unable to degrade DCA or pBS(ToMO) was lost.

Week 4

We re-did the ToMO trial with three major changes:

the volume of cells was increased from 50mL to 500mL before cells were spun down and incubated with DCA,
we introduced IPTG, to aid the expression of genes under the lac operon in pBS
we looked at both ToMO (toluene-o-xylene monooxygenase) and TOM (toluene ortho-monooxygenase.

Everything else remained the same, including:

using E. Coli transformed with pBBR as a control,
incubating cells with 1mM DCA.

Week 5

Chloride ASSAY: Another chloride assay was performed (ToMO, pBBR and TOM) to determine DCA metabolism efficacy. Chloride is release as a byproduct of DCA catabolism. ToMO best degraded DCA in this assay which no metabolism occurred in our control (pBBR).

Week 6

Attempt was made to PCR origin of replication from the plasmid, pBBR-mcs2 using primers iGEM5 and 6. Diagnostic gels of EcoR1 digested and undigested pBBR were performed. We did not observe the desired PCR product, possibly due to mispriming.

Week 7

Some competent Pseudomonas strutzeri strain Q cells were made for natural transformation of our constructs and tested for their growth. Reagents for plasmid preps and plates were prepared.

Week 8

We did a Gibson Assembly of our gBlocks and transformed them into E.coli (EPI300). We attempted to transform our parts into other E.coli hosts as well in the hope of getting better results.

Week 9

We found our transformation of the parts into TOP10 worked better than EPI300. We tested presence of dhlB in psb1c3 by incubating our clones in chloroacetate (pathway link) Based on a previous chloride assay we selected 10 colonies for another assay. The selections were made as follows: strong degraders (2, 37, 38 and 42, 64, 85), mid-range degradation (25 and 52) and without degradation (10 and 54). Results did not correspond between the two assays. This is perhaps because the first had a great deal more variance in the number of cells.

A plasmid prep was done but we found there was too much RNA contamination. The gel scan also suggests our gblock may have misassembled. It was repeated, this time with addition of RNase.

Week 10

Solutions were remade for plasmid preps and chloride assays to improve quality of stock solutions.

Another chloride assay using the clones that worked from the previous assay was done to measure Cl- production from breakdown of choloroacetate. This new assay includes a negative and positive biological controls. Another chloride assay was set up later in the week using the same clones but testing then in DCA and chloroacetate as substrates. Plasmid prep was done for all clones and patch plated (Lm-Cm).

Week 11

This week we did PCR of dhlB and ran them on gels for confirmation. Our Gibson Assembly products were transformed into E.coli cells Epi400. Another GA was set up and products were transformed into both E.coli TOP10 and Epi400.

Week 12

Gel run for dhlA showed the bands were consistent with the colour of the patched clones. However dhlB was not consistent so we tried screening for dhlB again from TOP10 transformants but this did not work either. We decided to use EPI300 transformants instead. Based on these gels we made plasmid preps from B110, B112, B114.

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+__NOTOC__
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+=='''Overview of What We Did'''==
+===Week 1===
+We performed a plasmid extraction of ToMo from E.coli (strain?). We then digested pBBR1-MCS2 using XbalI followed by gel of these samples including a marker and undigested pBBR. (link protocol- for plasmid prep, digest and gel)
+===Week 2===
+We used E.coli Epi300 to transform our two plasmids (pBBR and pBS-ToMO). Both contain Kanamycin resistant genes, allowing us to select for successfully transformed cells by growing them on LB-Km. E.coli (pSB-ToMO) was not growing well so transferred them to broth for further incubation.
+E.coli (pBBR) and E.coli (Psb-ToMO) were transferred and incubated in phosphate buffer solutions containing either 1mM DCA or no DCA overnight.
+===Week 3===
+We used the Gas Chromotrographer (GC) [hyperlink to chloride assay] to look at our two DCA treatments of E.coli. Both had similar results, which is not what we were hoping for. Two possible explaination for this  may be that either ToMO was unable to degrade DCA or pBS(ToMO) was lost.
+===Week 4===
+We re-did the ToMO trial with three major changes:
+*	the volume of cells was increased from 50mL to 500mL before cells were spun down and incubated with DCA,
+*	we introduced IPTG, to aid the expression of genes under the lac operon in pBS
+*	we looked at both ToMO (toluene-o-xylene monooxygenase) and TOM (toluene ortho-monooxygenase.
+Everything else remained the same, including:
+*	using E. Coli transformed with pBBR as a control,
+*	incubating cells with 1mM DCA.
+===Week 5===
+Chloride ASSAY: Another chloride assay was performed (ToMO, pBBR and TOM) to determine DCA metabolism efficacy. Chloride is release as a byproduct of DCA catabolism. ToMO best degraded DCA in this assay which no metabolism occurred in our control (pBBR).
+===Week 6===
+Attempt was made to PCR origin of replication from the plasmid, pBBR-mcs2 using primers iGEM5 and 6.
+Diagnostic gels of EcoR1 digested and undigested pBBR were performed. We did not observe the desired PCR product, possibly due to mispriming.
+===Week 7===
+Some competent Pseudomonas strutzeri strain Q cells were made for natural transformation of our constructs and tested for their growth.
+Reagents for plasmid preps and plates were prepared.
+===Week 8===
+We did a Gibson Assembly of our gBlocks and transformed them into E.coli (EPI300). We attempted to transform our parts into other E.coli hosts as well in the hope of getting better results.
+===Week 9===
+We found our transformation of the parts into TOP10 worked better than EPI300. We tested presence of dhlB in psb1c3 by incubating our clones in chloroacetate (pathway link)
+Based on a previous chloride assay we selected 10 colonies for another assay. The selections were made as follows: strong degraders (2, 37, 38 and 42, 64, 85), mid-range degradation (25 and 52) and without degradation (10 and 54). Results did not correspond between the two assays. This is perhaps because the first had a great deal more variance in the number of cells.
+A plasmid prep was done but we found there was too much RNA contamination. The gel scan also suggests our gblock may have misassembled. It was repeated, this time with addition of RNase.
+===Week 10===
+Solutions were remade for plasmid preps and chloride assays to improve quality of stock solutions.
+Another chloride assay using the clones that worked from the previous assay was done to measure Cl- production from breakdown of choloroacetate. This new assay includes a negative and positive biological controls.
+Another chloride assay was set up later in the week using the same clones but testing then in DCA and chloroacetate as substrates.
+Plasmid prep was done for all clones and patch plated (Lm-Cm).
+===Week 11===
+This week we did PCR of dhlB and ran them on gels for confirmation. Our Gibson Assembly products were transformed into E.coli cells Epi400. Another GA was set up and products were transformed into both E.coli TOP10 and Epi400.
+===Week 12===
+Gel run for dhlA showed the bands were consistent with the colour of the patched clones. However dhlB was not consistent so we tried screening for dhlB again from TOP10 transformants but this did not work either. We decided to use EPI300 transformants instead.
+Based on these gels we made plasmid preps from B110, B112, B114.
+===Week 13===
 {{Team:SydneyUni_Australia/Footer}}