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Lab Journal

Thursday 23/6

Lab equipment was prepared: Pipette tips, 1.5 ml tubes, toothpicks, SOC, LA with 100 µg/ml ampicillin/ 100 µg/ml spectinomycin (from 1988). Recipies are given in recipies section.

Friday 24/6

Biobricks were taken out from kit by resuspending them in sterile water followed by transformation to E.coli DH5 alpha. The biobricks that were taken out are given below:

Saturday 25/6

Plates with transformants were moved to fridge.

Sunday 26/6

Transformants were inoculated in 3 ml LB + proper antibiotics to prepare for isolation of plasmid.

Monday 27/6

Plasmids that contained P1, lambda pR, RFP and Lux biobricks were isolated by using miniprep kit from Promega.

Consentrations of the biobricks were measured and were as follows:

Transformants from Lux/RFP constructs were not red as expected. It was suggested that the phenotype could be explained by presence of TetR in the system. Since tetracycline (Tc) is an inhibitor of TetR we tried to grow the transformants in sublethal consentrations of Tc. E. coli with RFP were grown in 3 ml LB with 0, 0.1, 0.3, 0.6, 1.0, 1.5 and 100 µg/ml Tc.

Tuesday 28/6

It's Jon's birthday! Happy day!

Growing the bacteria in Tc did not result in red color of the culture

lambda pR was ligated to RFP backbone:

• RFP and Luc construct were cut with EcoRI and XbaI.
• lambda pR was cut with EcoRI og SpeI

Restriction digestion was done using [http://partsregistry.org/Help:Protocols/Restriction_Digest iGEM's protocol]

Since the restriction digestion gave unexpected fragments and we had not yet seen any pigmentation, we started looking for other designs that could give the result that we wanted. We came up with a different design that used pLac promoter and lacI repressor instead of the pTet/TetR system.

To start constructing the alternative system 3 new biobricks were transformed to E. coli:

Wednesday 29/6

Lambda Pr, RFP and Lux was cut:

• RFP with XbaI and PstI
• Lux with XbaI and PstI
• Lambda Pr with SpeI and PstI

The cutting gives RFP and Lux as insert and Lambda pR the backbone.

The restriction fragments were separated on agarose gel:

The restriction fragments for lambda pR were as expected but not for RFP or Lux.

Lambda pR was isolated from gel (2110 nt) by using DNA gel extraction kit.

DNA concentration of lambda pR was measured to 5.2 ng/µl

Biobricks that were transformed yesterday were inoculated in 3 ml LB with proper antibiotics to prepare for isolation of plasmids.

Anders ordered a biobrick that should contain LacI + RBS + double terminator from iGEM HQ: BBa_K292006

Thursday 30/6

Plasmid was isolated from lac-design biobricks. Concentration of DNA:

Kuttet lacP biobrick med SpeI og PstI. Mc ble kuttet med XbaI og PstI. Vil gi lacP backbone og Mc insert.

Forsøker direkte ligering av digest: 11µL H20

2µL backbone + 2µL insert 2µL T4 ligase buffer 1µL ligase

Mix, spin down, 30 min 16C, heat kill 20 min 80C. Transform 2µL. Selektere for røde kol.

Kjørte resten av prøven på gel. Kuttet ut MC insert (895 bp) og LacP backbone (2116 bp). Renset plasmid fra gelbitene med QIAquick Gel Extraction Kit. Målte cons med nanodrop

LacP: 8,5 ng/µL MC: 2,1 ng/µL

Transformerte den direkte ligeringen på amp og amp + IPTG plater. pluss kontroll.

Har undersøkt komposittbiobrikken RBS+LacI+dobbelt transkripsjonsstopp (BBa_K292006) for å se om denne er lik det vi hadde kommet til å lage selv fra RBS+LacI biobrikken (BBa_J24679) og den dobble transkripsjonsterminatoren (BBa_B0015). Har kuttet og ligert BBa_J24679 og BBa_B0015 i clone, og fått en biobrikke som har 100 % sekvenslikhet med BBa_K292006 bortsett fra på endene. Denne biobrikken er altså lik den vi ville laget selv, iallefall i teorien.

Har funnet nukleotidsekvensen på relA-genet, i tilfelle det blir behov for å lage primere til dette. Nukleotidsekvensen til relA har et kuttested for PstI midt i genet (basepar 1383-1388 med påsatt prefix), som kan bli et problem hvis man skal gjøre relA til en biobrick. Vi kan enten bruke partial digestion, eller indusere en punktmutasjon slik at CTGCAG → CTACAG eller CTGCAA uten at aminosyresekvensen forandres.

LacI-negativ E.coli:

For å få Lac-systemet vårt til å fungere som vi ønsker er vi mest sannsynlig avhengig av en LacI-negativ stamme. Etter mye søk kan det se ut som om Top10 er LacI-negativ. (http://partsregistry.org/Part:BBa_K177038). Denne får vi til promotorkarakterisering og bør bruke den til å teste konstruktet v

Sponsors

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NTNU Rector's Office Programme for Bioinformatics, NTNU