20.109 Spring 2007
Year of Graduation
Module 1: Genome Engineering
Module 1:Day 3
For next time:
Questions 1 and 2 are theoretical but they should help prepare you to interpret the results you will collect next time.
1. You have purchased some supercompetent bacteria that are provided at a transformation efficiency of 109 colony forming units/ug of DNA. You transform the cells with 1 ng of plasmid DNA and plate 1/1000th of the cells. How many colonies do you expect?
- [(10^9 CFU/ug)[(10^-9 ng)/(10^-6 ug)](1 ng)]/(1000)= number of colonies = 1000 colonies
Next you transform another aliquot of cells, also at 109 colony forming units/ug of DNA, with 2 μl of plasmid DNA. You spread 1/100th of the cells and find 50 colonies growing on the plate after 24 hours at 37°C. What is the concentration of plasmid?
- [(10^9 CFU/ug)[(X ug)/(2 ul)]]/(100)= 50 colonies
- X=1x10^-5 CFU/ml
To illustrate your understanding and the importance of the controls you performed today, please write a one-sentence interpretation for each of the following transformation outcomes. Outcome 1: no colonies on any plate.
- This could mean that we treated the bacteria with an antibiotic other that Kanamycin, one which they are not expected to already be resistant to. This could also mean that somehow we destroyed the integrity of all the fragile competent cells. This could also mean that there was an error in transforming the bacteria with phage DNA. If the phage DNA didn't enter the bacterial genome, then the bacteria would not be resistant to Kanamycin.
Outcome 2: thousands of colonies on all the plates.
- This could mean that we treated the bacteria with an antibiotic other that Kanamycin, but this time one which they are expected to already be resistant to. This could also just mean that the transformation of the plasmid was very efficient.
There may be more than one valid interpretation for some of the data (only one answer for each is required for the assignment).
Next time you will prepare DNA from four transformants and begin to characterize the plasmids in these bacteria. Using the plasmid map you drew last time, plan at least two restriction digests that will confirm the presence of the PCR insert. It will help to read the introduction for the next lab before you complete this part of the assignment. Be sure to predict the size of the fragments you expect when the plasmid does and doesn’t have the PCR insert. Also include reaction conditions such as buffer and temperature. Use the NEB website for details on various enzymes and reaction conditions (http://www.neb.com).
|Diagnostic digest 1||plasmid with insert||plasmid no insert|
|Buffer used||NEB #3||NEB #3|
|Predicted fragments||circular ~8700 bp||linear ~8700 bp|
|Diagnostic digest 2|
|Predicted fragments||linear ~8700||circular ~8700|
4. Based on the results of your plaque assay, what is the titer of each stock solution of phage? Please show your work.
- Results: 10^-6 K07 = 332 much smaller plaques
10^-6 E4 = 560 plaques
- K07 titer:
**(X PFU/ul)(10^-6 dilution)(10 ul)=332 PFU **X=3.32x10^7 PFU/ul
*E4 titer: **(X PFU/ul)(10^-6 dilution)(10 ul)=560 PFU **X=5.60x10^7 PFU/ul
If the plaques appeared different, please consider how the phage genomes differ (M13K07 is a "helper phage" while E4 is identical the the M13 genome except four glutamic acids are presented on the N-terminus of the p8 protein) and suggest how these differences might account for the differences in plaque morphology.
- The differences between the K07 and the E4 plaques was that E4 showed more and larger plaques. The added glutamic acids on the N-terminus (the displayed side) must somehow allow the E4 to incorporate more readily into bateria, which explains why there were more plaques. The other thing to explain is the size difference. E4 must be using up more of the bacteria's resources and retarding it's growth.
5. Read the article by Chan, Kosuri and Endy. "Refactoring bacteriophage T7" Nature/EMBO Molecular Systems Biology 13 September 2005 doi:10.1038/msb4100025 and News & Views. Come prepared to discuss this paper during lab next time. To guide your reading and test your understanding, try to answer the following questions (note: these questions are just to guide your reading and the answers do not have to be turned in):
from the Introduction: What is "refactoring" and what makes is T7 an attractive candidate for this approach? What experimental techniques give us "compenent level" understanding, i.e. allow us to attribute particular functions to particular sequences in the genome? How completely can "component level" understanding provide "system level" understanding? How predictive have computational and quantitative models for T7 behavior proven to be? What's important about predicting behavior? from the Results: What design principles were the authors pursuing? How well do these map to our class effort at M13 re-design? Was the entire T7 genome refactored? What techniques were used to verify the refactoring? What techniques were used to evaluate it? from the Discussion: How do the authors' findings extend knowledge of T7 biology? Does T7.1 resolve disagreements between model-based behavior predictions and those that are observed though experimental approaches? Could nature have produced the T7 phage that now exists in the Endy lab in Building 68? What's next for this phage?
6. Decide if you want to be a "Discoverer" or a "Designer" for the M13 refactoring project. Discoverers will work to identify, define, refine, and perhaps erase regions of the wild-type M13 genome. Designers will work to engineer new features into the genome in support of applications (e.g., what Professor Belcher discussed in class 15 Feb). Note: it would be great to have both types of folks (i.e., discoverers & designers). Please state what type of work you want to do here.
- Designer (unless Discoverers are needed)
7. If you have not done so already, register for an account on the Registry of Standard Biological Parts; join the 20.109 spring '07 group.
8. On the Parts Registry, annotate BBa_M1307 by adding two "features" namely a genetic element of your choosing (an ORF, promoter, or RBS) and one single-cutter restriction endonuclease site. You'll have to choose a genetic element and restriction site that hasn't been added by one of your classmates. Adding a feature requires that you
login to the registry pull up BBa_M1307 using the search window on the lefthand navigation bar click on the "Hard Information" link that is in the lefthand navigation bar click on the edit tab that is at the top of the page (near the "article" and "discussion" tabs) click on the edit link that is on the right side of the page above the "sequence and features" box click the "add a feature" link that is near the features box fill in the "type" "label" "start" and "stop" details for the feature you're adding Note what you added to the annotation next to your name here. Hint! You can find a table listing the Registry Part numbers for M13 genes, promoters, and RBSs here. Use the part numbers to find the DNA sequence information of the genetic elements and then use this information to annotate the full M13K07 sequence. Hint #2! Remember that you can find a listing of single cutters here.
- added g4 RBS, g11 RBS, and Drd1 site
Module 1:Day 4
- Prepare a table with the results of your ligations and transformations. Calculate your transformation efficiency (# colonies/μg plasmid DNA) based on the transformation you performed with M13K07. In three or four sentences, interpret the ligation results.
|Tube||Transformation||Add||Expected Number of Colonies||Actual Number of Colonies||Transformation Efficiency (# colonies/ug DNA)|
|1||positive control plasmid||1 μl (5 ng) of M13K07 DNA||many||1008||210600|
|2||bkb, no ligase||5 μl||0||0||N/A|
|3||bkb, plus ligase||5 μl||0||0||N/A|
|4||bkb+insert, plus ligase||5 μl||many||4||N/A|
All of our controls were confirmed so we have confirmed that the cells are competent and the plasmid DNA can infect them (ligation 1), that there was no errant uncut plasmid left (ligation 2), and that the backbone didn't just reconnect with itself (ligation 3). Ligation 4 is our actual experimental data. We expected more colonies than we got, so that means there was some error in the procedure that kept our transform efficiency down. We ran a gel to inspect the viability and success of the fourth ligation, but were unable to see the picture in the scanned version. Hopefully we will be able to analyze our results better in lab tomorrow.
- Refactor the M13K07 genome from the unique HpaI site in gene II through the unique BamHI site in gene III, guided by the algorithm described in the Refactoring T7 paper Supplementary Figure 1. Start by making a new part of this region on the Registry of Standard Biological Parts (note: allowed names for new parts from our class must fall between BBa_M30000 to BBa_M31999) and then fully annotating the M13K07 sequence across this region. Include component descriptions for all genetically encoded functions. Be sure to separately specify any new parts(s) that can be used to define the M13.1 sequence. If there are particular aspects of the M13K07 sequence that cannot be edited, start a table to list these, describing the design impass in sufficient detail that someone unfamiliar with our work might understand enough to help. Finally, describe the refactoring work you've done in paragraph and table form, ideally working from your wiki user page, to hand in next time. The table should include information about your annotated M13K07 entry as well as any new part(s) you've designed.
I plan to unpack all the genes from inside one another with create discrete units of them with a few exceptions. I will leave gene X (including it's promoter) packed into gene II. The only changes I will make to gene II are to weaken the promoter and RBS binding sites for gV that are within gII. The rest of the genes will be separate from eachother and in a format as follows: [...promoter...(around 20 bp break)...RBS, ORF...] Because gene VII has no promoter, I will put in a copy of the promoter for gene V (where gene VII was packed). Everywhere where I have made a copy of a promoter or RBS outside of a gene, I will weaken the original that lies within the gene. Also, between each of the units, I will insert restriction sites for easy manipulation.
|Item||Change||Original Location (in M13K07)||New Location (in BBa_M31515)||Basic Part ID Number|
|gene II ORF||weaken prom gV and RBS gV, includes HpaI site and gene X promoter, RBS, and ORF||8268-831||BBa_M3196|
|EagI||new restriction site||none||BBa_M3195|
|BlpI||new restriction site||none|
|promoter gV||add new copy||129-148|
|RBS gV||add new copy||827-842|
|ORF gV||weaken RBS gVII||843-1106|
|BlpI||new restriction site||none|
|BsiWI||new restriction site||none|
|promoter gV||new copy to promote gVII||843-1106|
|RBS gVII||new copy||1092-1107|
|ORF gVII||weaken promoter gVIII||1108-1209|
|BsiWI||new restriction site||none|
|KasI||new restriction site||none|
|promoter gVIII||new copy||1155-1201|
|RBS gVIII||new copy||1155-1201|
|ORF gVIII||weaken gIII promoter||1301-1522|
|KasI||new restriction site||none|
|NarI||new restriction site||none|
|promoter gIII||add new copy||1500-1547|
|RBS gIII||new copy||1579-2853|
- to weaken an RBS: go 9-11 bp's behind start codon, and change A's and G's to T's and C's.