Team:Cambridge/Experiments/Assembly of Reflectin Constructs

From 2011.igem.org

(Difference between revisions)
(→Diagnostics)
(→Results)
Line 99: Line 99:
===Results===
===Results===
-
After overnight incubation at 37°C of transformed ''E.coli'' cells we could not see distinct colonies on any of our plates in the morning. Thus, we decided to conduct a series of tests to identify the cause of the failure. The proposed sources of error included:
+
After overnight incubation at 37°C of transformed ''E.coli'' cells, we could see colonies on only some of our plates. We examined successful plates under a fluorescence miscroscope.
 +
 
 +
{|
 +
|'''GA1'''
 +
|'''GA2'''
 +
|'''GA3'''
 +
|'''GA4'''
 +
|-
 +
|
 +
 
 +
 
 +
 
 +
Thus, we decided to conduct a series of tests to identify the cause of the failure. The proposed sources of error included:
*Low efficiency of DNA gel extraction
*Low efficiency of DNA gel extraction
*Unsuccessful Gibson Assembly
*Unsuccessful Gibson Assembly

Revision as of 13:45, 26 August 2011

Loading...
OVERVIEW
home

This is a placeholder. We should fill it in.

Contents

Construct Design

Primer Design

We should mention expected lengths of products here.

Assembly: first attempt

PCR

In the first round of PCR, we amplified fragments required for the assembly of GA1, GA2, GA3 and GA4 constructs.

Hold 95°C 2 min
Cycling Denaturing 95°C 10 s
Annealing 55°C 20 s
Elongation 72°C 150 s

We decided to use the 55°C annealing temperaure, although the predicted temperature for most primers is 5-10°C higher, because of a low annealing temperature of the VF2 primer.

  • Primers and template DNA provided by our supervisor Paul served as a positive control, but eventually we did not detect any products on the gels.


The pictures below present result of gel electrophoresis of PCR products.

  • In most cases position of a band matches the expected length of DNA fragment. The only exception are GA1-2 and GA3-2 products. According to the position on the gel the length of these DNA fragments is 4-5kb, whereas the predicted length is 3.5kb. Our hypothesis is that we were provided [http://partsregistry.org/Part:pSB1AK3 pSB1AK3] backbone instead of [http://partsregistry.org/Part:pSB1A3 pSB1A3]backbone.
  • For GA1-1, GA2-1, GA3-1 and GA4-1 we obtained two bands: 1000bp and 400bp, with the latter resulting from non-specific priming most probably. We extracted the two bands for GA1-1, GA2-1 and GA4-1 products, labelling the 1000kb and 400bp fragments GAX-1a and GAX1-b respectively.
  • The molecular weight marker that we used in all gels is HyperLadder I, which produces regularly spaced bands ranging from 200 to 10,000bp.

For the gel extraction of DNA we followed the protocol, assuming that one slice of gel is 100μl.

Gibson Assembly

We conducted Gibson Assembly of GA1, GA2, GA3 and GA4 constructs according to [Team:Cambridge/Protocols/Gibson_Assembly#Practice | the protocol]. The volumes of Master Mix and solutions of amplified DNA were the following:

GA1 GA2 GA3 GA4
15µl Master Mix 15µl Master Mix 15µl Master Mix 15µl Master Mix
2.5µl GA1-1a 1.67µl GA2-1a 2.5µl GA3-1 1.67µl GA4-1a
2.5µl GA1-2 1.67µl GA2-2 2.5µl GA3-2 1.67µl GA4-2
1.67µl GA2-3 1.67µl GA4-3

Transformation

We transformed competent E.coli cells according to [Team:Cambridge/Protocols/Transformation_of_E.Coli | the following protocol]. We cultured each class of transformants on four different plates.

GA1 and GA3 GA2 and GA4
Plate 1 (10μl of cell suspension) LB + ampicillin LB + kanamycin
Plate 2 (100μl of cell suspension) LB + ampicillin LB + kanamycin
Plate 3 (10μl of cell suspension) LB + ampicillin + arabinose LB + kanamycin + arabinose
Plate 4 (100μl of cell suspension) LB + ampicillin + arabinose LB + kanamycin + arabinose

Results

After overnight incubation at 37°C of transformed E.coli cells, we could see colonies on only some of our plates. We examined successful plates under a fluorescence miscroscope.

GA1 GA2 GA3 GA4


Thus, we decided to conduct a series of tests to identify the cause of the failure. The proposed sources of error included:

  • Low efficiency of DNA gel extraction
  • Unsuccessful Gibson Assembly
  • Low viability of competent E.coli cells

Diagnostics

In order to check if gel extraction of DNA was successful, we ran a 1% agarose gel with samples of purified products of the initial PCR reactions. Although we could see distinct, fairly thick bands on the first gels, from which components of Gibson constructs were purified, bands on the diagnostic gel are very faint, even missing in some lanes. This indicates that the yield of our extraction procedure was very low.

Assembly: second attempt

PCR

In the second round of PCR, we amplified fragments required for the assembly of GA1, GA2, GA3, GA4 as well as GA5 and GA6 constructs.

We ran PCR products on a 1% agarose gel to separate amplified products from template DNA and primers, as well as to check how efficient and specific the amplification process was.

  • All components of GA5 and GA6 constructs produced clear fairly thick bands with positions matching expected lengths. No non-specific bands on GA5-1 (Reflectin A2) and GA6-1 (Reflectin 1B) lanes were observed.
  • The molecular weight marker that we used in all gels is HyperLadder I, which produces regularly spaced bands ranging from 200 to 10,000bp.

Gibson Assembly

Transformation

Results

What next?
Retrieved from "http://2011.igem.org/Team:Cambridge/Experiments/Assembly_of_Reflectin_Constructs"