Part:BBa_K381001:Experience

Applications of BBa_K381001

Nitrate and Nitrite sensitive promoter PyeaR with a GFP coding device and strong RBS to create a nitrate-sensitive system which signals through expression of GFP.

User Reviews

UNIQ8f184cd556e2ac0e-partinfo-00000000-QINU

••••• EliasGrimaldo

The part work well under cold shock stress (20°C to 30°C) of 10-12 hr.

UNIQ8f184cd556e2ac0e-partinfo-00000002-QINU

iGEM11_UTP-Panama Experience: Cold Shock to BBa_K381001

Objectives:
1. Test the BioBrick operation to different substrates and temperatures, using KNO3 40 mM:
a.Substrates:
i.LB
ii.Minimal essential medium (MM)
iii.Saline solution (SS)
iv.Lunar Land (not used)
b.Temperatures:
i.8°C
ii.X°C (not used)
iii.23°C
iv.37°C
2. Improve the characterization of the BioBrick tested in other substrates to study the performance of the promoter.
3. Define the Biobrick promoter strength at the temperatures and selected substrates.

Methodology
We chose 4 temperatures: ´

• 5-10°C
  
• 20-30°C
  
• 37°C
  

These temperatures were selected so we could evaluate the response to cold shock in various ranges of our BioBrick. Since the lab equipments we had available couldn’t reach temperatures between 11-20°C this temperature was not used during the experience. Finally, the lab equipment allowed us to make the experiments in: 37°C, 23°C and 8°C.
We also chose 3 different Growth Media:

• Saline Solution
  
• Liquid LB
  
• Minimal Media
  

These growth medias where selected to evaluate the response of our BioBricks in different metabolic pathways. According to the characterization made by Team Edinburgh 2009, the Pyear promoter (BBa_K216005) used by Bristol 2010, works best in minimal media and complex media with a nitrate concentration of 40mM. Using this reference, we added 40mM of KNO3 to every growth media in the different temperatures for Bristol 2010 - BBa_K381001 and UTP-Panama 2011 – BBa_K672000.
We prepared 9 15mL Falcon Tubes, these tubes contained:

• First 3 (one for each temperature): 10mL of Saline Solution + a colony of the BioBrick + 40mM of KNO3.
• Second 3 (one for each temperature): 10mL of Minimal Media + a colony of the BioBrick BioBrick + 40mM of KNO3.
• Lat 3 (one for each temperature): 10mL of Liquid Luria Broth + a colony of the BioBrick BioBrick + 40mM of KNO3.
  

RESULTS

iGEM11_OUC Experience

Applications of BBa_K381001

From the beginning, we use the linearized plasmid backbone pSB1C3 in the 2011 spring DNA distribution. But the standardization with them all end up with failures.

Then, we began to seek for a new plasmid backbone. We found the K381001 in pSB1C3. If we cut the pSB1C3 with EcoR I and Pst I, we could get pSB1C3 plasmid backbone for standardization.

User Reviews

UNIQ8f184cd556e2ac0e-partinfo-00000003-QINU

••••• DonQuixia

When we use the pSB1C3 for standardization, we found that the pSB1C3 have the right antibiotic resistance but a wrong DNA length.

UNIQ8f184cd556e2ac0e-partinfo-00000005-QINU

The verification

The pSB1C3 is supposed to be 2049bp, however, it appears around the marker 3000bp. We have the pSB1C3 in K381001 sequenced and found its total length is 2944bp, and its total sequence is as below,

Through sequence analysis, we could find that the base in blue have a 99% homology with the right pSB1C3. It lacks a C and a A in the beginning. In the end, it has a 71 base homology with the right pSB1C3. The other sequence left do not accord with the right pSB1C3.