"
Team:NTNU Trondheim/Journal
From 2011.igem.org
(→Wednesday 29/6) |
(→Lab Journal) |
||
| Line 143: | Line 143: | ||
Anders ordered a biobrick that should contain LacI + RBS + double terminator from iGEM HQ: '''BBa_K292006''' | Anders ordered a biobrick that should contain LacI + RBS + double terminator from iGEM HQ: '''BBa_K292006''' | ||
| + | |||
| + | ==Thursday 30/6== | ||
| + | |||
{{:Team:NTNU_Trondheim/NTNU_footer}} | {{:Team:NTNU_Trondheim/NTNU_footer}} | ||
Revision as of 11:15, 5 July 2011
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:
| Abbreviation | Name | Part number | Resistance |
|---|---|---|---|
| P1 | rrnB P1 | BBa_K112118 | Spec |
| lambda pR | lambda pR mod | BBa_R0051 | Amp |
| RFP | TetR + p(TetR)+RFP | BBa_K092600 | Amp |
| Lux | TetR + p(TetR)+RFP+luxI | BBa_K092700 | Amp |
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:
| Biobrick | Concentration (ng/µl) |
|---|---|
| P1 | 98.4 |
| lamda pR | 43.8 |
| RFP | 141.8 |
| Lux | 62.8 |
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:
| Abbreviation | Name | Part number |
|---|---|---|
| LacI | LacI + RBS | BBa_J24679 |
| pLac | Lac promoter hybrid | BBa_R0011 |
| mCherry | mCherry + RBS + term | BBa_J06702 |
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
