8 - generating BIMs

Question 1

what is the plaque assay efficiency

Answer 1

number of plaque forming units is always lower than the actual counts of viral aprticles made microscopically

have more viral particle sin your sample than waht you calculate

there could be a chance that 2 bacteriophages infect 1 bacteria

Question 2

why are BIMs resistant to phage infection?

Answer 2

CRISPR: Clustered regularly interpsaced short palindromic repeats
* major antiviral defence system in some Bacteria(70%) & Archaea(90%)
* adaptive immunity (memory) in bacteria – specific to virus they can recognize because of spacers
* seeks & destroys foreign/invading DNA in bacteria

Question 3

what are the different ways abcteria can prevent phage infection?

Answer 3

• alter the receptyor the bacteriophage uses to attach to the host
• use RE cut up foreigh DNA/nucleotides
• methylate viral DNA/RNa, no more repliaction
• bacteria go under programmed celld death
  * CRISPR-Cas system: specifically recognizes the foreign DNA & can degrade or silence it

Question 4

what is the CRISPR-Cas system?

Answer 4

CRISPR array:
* have interspaced repeats (20-50 nt): same size & same sequence, interspaced between unique spacers
* spacer: pieces of foreign DNA (memory banks): unique sequences of foreign DNA
1. CRISPR array is transcribed into long piece of CRISPR RNA
2. Cas genes are transcribed & translated into Cas proteins
3. Cas proteins cut the CRISPR RNA = each piece has 1 of the unique spacers & forms a copmplex with cas Protein
4. if there viral/oincvading DNA, Cas + spacer scan throught eh foreign DNA
5. if theres complementarity to the sapcer, the Cas protein will chop up the invading DNA

2 sections: CRISPR array & Cas proteins

Cas: CRISPR associated

Question 5

how can Cas protein differentiate between self & non-self?

Answer 5

PAM sequence

Question 6

what is the CRISPR defense system agains viruseS?

Answer 6

when a phage infects its DNA:
* a sequence PAM (Protospacer adjacent motifs: 5-6nt) = determines non-self, recognized by Cas proteins, essential for cutting
* protospacer: part of viral DNA near the PAM sequence
* protospacer becomes spacer: when Cas protein takes protospacer into the CRISPR array = immunization
* interference = if the bacteria encounters the same viral DNA, the antiviral complexes with defferent spoacers scan the viral DNA and recognize the PAM sequence as non-self if theres complementarity, the Cas rpoteins will silence it

immunization

Cas protein recognize PAM sequence and knows its not the bacterial DNA, it is non-self

Question 7

how does the CRISPR/Cas9 system?

Answer 7

1. Cas proteins 1 and 2 recognize & integrate a piece of foreign DNA as a spacer into the CRISPR array at the 3’ end of the leader sequence (at the front)
2. CRISPR array is transcribed into long pre-CRISPR RNA (pre-crRNA)
3. Trans-activating CRISPR RNAs (tracrRNA = noncoding), crRNA (pre-crRNA chopped up by RNAse III) & Cas 9 form a complex that is cut = an antiviral complex
4. Scans dsDNA for the presence of PAM & complementary binding to crRNA
5. Cas9 cuts (silences) the invadsing DNA

type 2A in S. thermophilus

Question 8

CRISPR-Cas9 System in S. thermophilus

CRISPR loci in S. thermophilus

Answer 8

S. thermophilus has mainly 2 Type II-A systems are active CRISPR-Cas Systems providing immunity
* Loci 1 (CR1) and 3 (CR3)
* PAM sequence recognized by Cas proteins for CR1: NNAGAAW
* PAM sequence recognized by Cas proteins for CR3: NGGNG

when a new spacer is added, its added at the 3’end of the leader seqeunce = addtional 60/66 nt on the array
* because theres also repeats that are interspaced

N = any nucleotide, W=A or T

there is 4 distinctive CRISPR-Cas system identified & over 90% of sapcer acquisition are found in CR1 locus

Question 9

CRISPR-Cas9 System in S. thermophilus

DGCC7710 vs DGCC7710 + pNZCR3

Answer 9

DGCC7710 strain of S. thermophilus = WT
* found in its natural, non mutatesd (unchanged0 form

DGCC7710 + pNZCR3 contains plasmid that increases BIM generation > 400 fold
* a plasmid with CR3 proptospacer and the corresponding OPAM from phage 2972 genome (also a chloramphenicol resistance gene)

this strain of bacteria we are using is WT

Question 10

CRISPR-Cas9 System in S. thermophilus

what is DGCC7710 + pNZCR3

Answer 10

• for CIRSPR to work, spacer must already be present
• spacers can be acquired by: Inactiated viruses & Plasmids
  -give abcteria enough time to immunized and not get lysed.
• pNZCR3 contains the protospacer & PAM sequence of phage 2972 that can be acquired into CR3
  -this plasmid is foreign DNA for the bacteria, so CRISPR-Cas system scans it and finds the PAM and takes the protospacer acquires it as spacer, gives enough time to acquire the spacer, get immunizes and when this bacteria that contains the plasmid is challenged with phage = generate more BIMs

Question 11

what are Cas proteins responsible for?

Answer 11

Finding PAM sequence & cutting the protospacer form foreign DNA

Question 12

what forms the antiviral complex in the CRISPR-Cas9 system?

Answer 12

crRNA , tracrRNA & Cas9

Question 13

summary

what is the CRISPR-Cas 9 system?

Answer 13

it provides a memory bank to bacteria
* leader sequence follwoed by repeats, then spacer
* pre-crRNA
* tracrRNA, crRNA & CAs9 compoelx scan dsDNA for the presence of PAM & cut invading DNA

Question 14

summary

what is Protospacer & spacer

Answer 14

• protospacer = viral DNA near PAM sequence in viral genome
• spacer = piece of foreign DNA in CRISPR array

Question 15

summary

what does pNZCR3 contain?

Answer 15

it contians the PAM sequence & protospacer that is acquired in CRISPR3 array

Question 16

quiz - T or F

(A) if your plaque assay has clear lysis and onlya. few colonies = BIM
(B) the ~66 bp addition in the CRISPR array is due t the length of the integrated spacer

Answer 16

T
F

Question 17

quiz

whats the difference between spacer and protospacer?

Answer 17

protospacer is found on the viral DNA and the spacer is found in the ACRISPR array

Question 18

quiz

what is 1 method you can use to increase the number of BIMs besidfes challenfin WET S/ thermopghilus with pahge infection?

Answer 18

by adding PnZCR3, a plasmid that contains PAM + Protospacers and chloroamphenical resistance = can increase BIM generation by >400 folds

Question 19

quiz

what is the purpose of PAM sequence?

Answer 19

to reocgnize the plasmid as non-self when Cas protein recognize it

Question 20

quiz

steps of CRIDPR-Cas9 system:

Answer 20

1. new spacer acquired into CRISPR array
2. Pre-crRNA synthesized
3. antiviral complex scans foreign dsDNA fopr complementary binding to crRNA
4. Cas9 protein silences invading DNA, reuslting in interference of phage infection

Question 21

review

CRISPR-Cas system: how many types, which one do we study, main components?

Answer 21

1. 6 types
2. we study type 2, the most studied system, ususally found in Streptococcus pyrogenes (only a single endonucelase)
3. CRIDPR array, Cas protein, Guide RNA